US10239917B2 - Method for identifying novel minor histocompatibility antigens - Google Patents

Method for identifying novel minor histocompatibility antigens Download PDF

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US10239917B2
US10239917B2 US15/299,387 US201615299387A US10239917B2 US 10239917 B2 US10239917 B2 US 10239917B2 US 201615299387 A US201615299387 A US 201615299387A US 10239917 B2 US10239917 B2 US 10239917B2
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Claude Perreault
Pierre Thibault
Sébastien Lemieux
Diana Paola Granados
Sriranganadane Dev
Mohamed Tariq Daouda
Olivier Caron-Lizotte
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Universite de Montreal
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    • G16B30/10—Sequence alignment; Homology search
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    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
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    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70539—MHC-molecules, e.g. HLA-molecules
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    • G16B20/50—Mutagenesis

Definitions

  • the present invention generally relates to histocompatibility antigens, and more specifically to minor histocompatibility antigens (MiHAs), identification and use thereof.
  • MiHAs minor histocompatibility antigens
  • Histocompatibility antigens are a group of cell membrane alloantigens that are recognized by T lymphocytes and thereby initiate graft rejection or graft-versus-host disease (GVHD) following transplantation (1).
  • MHC major histocompatibility complex
  • MiHA minor histocompatibility antigens
  • MHC antigens are transmembrane glycoproteins encoded by closely linked polymorphic loci located on chromosome 6 in humans. Their primary role is to bind endogenous and exogenous peptides that are scrutinized by T cells. MHC (or HLA) molecules present thousands of peptides at the surface of human cells (4;5).
  • MHC-associated peptides are referred to as the immunopeptidome and they originate from proteasomal processing and further processing of endogenous proteins (6-8).
  • the immunopeptidome of identical twins also referred to as syngeneic subjects
  • MAPs present on cells from HLA-identical non-syngeneic subjects are classified into two categories: i) invariant MAPs which are present in all subjects with a given HLA type, and ii) MiHAs which are MAPs present in some but absent in other subjects (9).
  • T cells When T cells are transplanted into an MHC-identical host, they react promptly and specifically to what they see as non-self: host-specific MiHAs.
  • MiHAs are essentially genetic polymorphisms that are immunogenic for T cells. MiHAs are a consequence of any form of accumulated genetic variation that translates to differential MAP display (3;9-13).
  • ATCI T-cell immunotherapy
  • the term “ATCI” refers to transfusion of T lymphocytes that may come from different types of donors: the patient (autologous), a genetically-identical twin (syngeneic), or a non-identical donor (allogeneic).
  • AHCT allogeneic hematopoietic cell transplantation
  • the graft-versus-tumor (GVT) effect induced by allogeneic AHCT is due mainly to T-cell responses against host MiHAs: GVT is abrogated if the donor is an identical twin (no MiHA differences with the recipient) or if the graft is T-cell depleted (20;23). More than 200,000 individuals treated for hematological malignancies owe their life to the MiHA-dependent GVT effect which represents the most striking evidence of the ability of the human immune system to eradicate neoplasias (18;24-28). Though the allogeneic GVT effect is being used essentially to treat patients with hematologic malignancies, preliminary evidence suggests that it may be also effective for the treatment of solid tumors (29-33).
  • MiHA-based ATCI is limited to “conventional” AHCT, that is, injection of hematopoietic cells from an allogeneic HLA-matched donor.
  • Such unselective injection of allogeneic lymphocytes is a very rudimentary form of MiHA-targeted therapy.
  • unselected allogeneic T cells react against a multitude of host MiHAs and thereby induce GVHD in 60% of recipients. GVHD is always incapacitating and frequently lethal (34-38).
  • CTLs cytotoxic T lymphocytes
  • MS mass spectrometry
  • MiHA discovery is made one by one and depends on the availability of a CTL line.
  • MiHAs that have been eluted from living cells and identified by MS can be considered to be validated (direct identification). In the other cases (indirect identification), uncertainty remains as to the exact structure of MiHAs naturally presented at the cell surface (an important criterion for MiHA-targeted immunotherapy).
  • T cells are eminently cross-reactive and can recognize more than one peptide (68); ii) bioinformatic tools used for identification of MAPs in general and MiHAs in particular do not have sufficient reliability to replace direct proteomic identification (69-71).
  • the present invention provides a method of identifying a minor histocompatibility antigen (MiHA) candidate, the method comprising: (a) isolating and sequencing MHC-associated peptides (MAPs) in a first cell sample from a first subject; (b) performing a whole transcriptome and/or exome sequencing on a second cell sample obtained from said first subject; (c) comparing the sequenced whole transcriptome and/or exome to a reference genome to identify single nucleotide variations (SNVs) between the transcriptome and/or exome of said first subject and the reference genome; (d) in silico translating the sequences containing the identified SNVs to identify peptide sequences comprising at least one non-synonymous mutation caused by said SNVs; (e) comparing the sequences of the MAPs isolated in (a) with the peptide sequences identified in (d); and (f) identifying a MiHA candidate based on said comparison.
  • MiHA minor histocompatibility antigen
  • the present invention provides a method of identifying a minor histocompatibility antigen (MiHA) candidate, the method comprising: (a) isolating and sequencing MHC-associated peptides (MAPs) in a first cell sample from a first and second subjects, wherein said first and second subjects are human leukocyte antigen (HLA)-matched; (b) performing a whole transcriptome and/or exome sequencing on a second cell sample obtained from said first and second subjects; (c) comparing the sequenced whole transcriptomes and/or exomes to identify single nucleotide variations (SNVs) between the transcriptomes and/or exomes of said first and second subjects; (d) in silico translating the sequences containing the identified SNVs to identify peptide sequences comprising at least one non-synonymous mutation caused by said SNVs; (e) comparing the sequences of the MAPs isolated in (a) with the peptide sequences identified in (d); and (f) identifying a MiHA
  • the above-mentioned MiHA candidate is a MAP whose sequence comprises at least one mutation relative to the corresponding sequence translated from the reference genome.
  • the above-mentioned MiHA candidate is a MAP present in the first cell sample from said first subject but absent from the first cell sample from said second subject.
  • the above-mentioned reference genome is the Genome Reference Consortium Human Build 37 (GRCh37).
  • the above-mentioned first and/or second cell sample is a peripheral blood cell sample.
  • the above-mentioned peripheral blood cell sample an immortalized peripheral blood cell sample.
  • the above-mentioned immortalized peripheral blood cell sample is an Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell line.
  • EBV Epstein-Barr virus
  • the above-mentioned isolating MAPs comprises (i) releasing said MAPs from said cell sample by mild acid treatment; and (ii) subjecting the released MAPs to chromatography.
  • the above-mentioned method further comprises filtering the released peptides with a size exclusion column prior to said chromatography.
  • the above-mentioned size exclusion column has a cut-off of about 3000 Da.
  • the above-mentioned chromatography is cation exchange chromatography.
  • the above-mentioned peptide sequences of (d) have a length of 12 amino acids or less. In a further embodiment, the above-mentioned peptide sequences of (d) have a length of 8 to 11 amino acids.
  • the above-mentioned comparing comprises subjecting the MAPs isolated in (a) to mass spectrometry and comparing the MS spectra obtained with the peptide sequences identified in (d).
  • the above-mentioned method further comprises determining the binding of the MiHA candidate identified in (f) to a major histocompatibility complex (MHC) class I molecule.
  • MHC major histocompatibility complex
  • the present invention provides a peptide of 50 amino acids or less comprising the sequence (I)
  • Z 1 -X 1 LQEKFX 2 SX 3 -Z 2 (I) wherein Z 1 is an amino terminal modifying group or is absent; X 1 is a sequence of 1 to 43 amino acids or is absent; X 2 is L or S; X 3 is a sequence of 1 to 43 amino acids or is absent; and Z 2 is a carboxy terminal modifying group or is absent.
  • X 1 is an acidic amino acid, in a further embodiment glutamic acid (E).
  • X 3 is an amino acid, in a further embodiment a hydrophobic amino acid, more particularly leucine (L).
  • X 2 is L.
  • X 2 is S.
  • the peptide comprises the sequence ELQEKFLSL (SEQ ID NO: 15), in a further embodiment the peptide is ELQEKFLSL (SEQ ID NO: 15). In another embodiment, the peptide comprises the sequence ELQEKFSSL (SEQ ID NO: 16), in a further embodiment the peptide is ELQEKFSSL (SEQ ID NO: 16).
  • the present invention provides a method of treating cancer, said method comprising administering to a subject in need thereof an effective amount of CD8 T lymphocytes recognizing a MHC class I molecule of the HLA-B*0801 allele loaded with the above-mentioned peptide (I).
  • the above-mentioned method further comprises determining whether said subject expresses a CENPF nucleic acid comprising a T or a C at a position corresponding to nucleotide 4409 in the nucleic acid sequence of human CENPF ( FIGS.
  • the above-mentioned determining comprises sequencing a CENPF nucleic acid.
  • the above-mentioned CD8 T lymphocytes are in vitro expanded CD8 T lymphocytes.
  • the above-mentioned method further comprises: (i) if said subject is the subject of (a), culturing CD8 T lymphocytes from a second subject comprising a C at a position corresponding to nucleotide 4409 in the nucleic acid sequence of human CENPF and/or a CENPF polypeptide comprising a serine residue at a position corresponding to residue 1412 in the protein sequence of human CENPF in the presence of cells expressing a MHC class I molecule of the HLA-B*0801 allele loaded with the above-mentioned peptide (I), wherein X 2 is L in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (ii) if said subject is the subject of (b), culturing CD8 T lymphocytes from a second subject comprising a T at a position corresponding to nucleotide 4409 in the nucleic acid sequence of human CENPF and/or a CENPF polypeptide comprising a le
  • the above-mentioned subject is an allogeneic stem cell transplantation (ASCT) recipient.
  • ASCT allogeneic stem cell transplantation
  • the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising: (a) determining whether a candidate donor expresses a CENPF nucleic acid comprising a T or a C at a position corresponding to nucleotide 4409 in the nucleic acid sequence of human CENPF ( FIGS. 1A to 1D , NCBI Reference Sequence: NM_016343.3), and/or a CENPF polypeptide comprising a leucine or serine residue at a position corresponding to residue 1412 in the protein sequence of human CENPF ( FIG.
  • the present invention provides a peptide of 50 amino acids or less comprising the sequence (II)
  • Z 1 -X 4 ELDX 5 VFQX 6 X 7 -Z 2 (II) wherein Z 1 is an amino terminal modifying group or is absent; X 4 is a sequence of 1 to 43 amino acids or is absent; X 5 is G or R; X 6 is an amino acid or is absent; X 7 is a sequence of 1 to 43 amino acids or is absent; and Z 2 is a carboxy terminal modifying group or is absent.
  • X 4 is glutamine (Q).
  • X 6 is an amino acid, in a further embodiment a basic amino acid, more particularly lysine (K).
  • X 7 is an amino acid, in a further embodiment leucine (L).
  • X 5 is G.
  • X 5 is R.
  • the peptide comprises the sequence QELDGVFQKL (SEQ ID NO:17).
  • the peptide is QELDGVFQKL (SEQ ID NO:17).
  • the peptide comprises the sequence QELDRVFQKL (SEQ ID NO:18).
  • the peptide is QELDRVFQKL (SEQ ID NO:18).
  • the present invention provides a method of treating cancer, said method comprising administering to a subject in need thereof an effective amount of CD8 T lymphocytes recognizing a MHC class I molecule of the HLA-B*4403 allele loaded with the above-mentioned peptide (II).
  • the above-mentioned method further comprises determining whether said subject expresses a ZWINT nucleic acid comprising an A or a G at a position corresponding to nucleotide 596 in the nucleic acid sequence of human ZWINT ( FIG. 2A , NCBI Reference Sequence: NM_007057.3), and/or a ZWINT polypeptide comprising a arginine or glycine residue at a position corresponding to residue 187 in the protein sequence of human ZWINT ( FIG.
  • the above-mentioned determining comprises sequencing a human ZWINT nucleic acid.
  • the above-mentioned CD8 T lymphocytes are in vitro expanded CD8 T lymphocytes.
  • the above-mentioned method further comprises: (i) if said subject is the subject of (a), culturing CD8 T lymphocytes from a second subject comprising a G at a position corresponding to nucleotide 596 in the nucleic acid sequence of human ZWINT and/or a ZWINT polypeptide comprising a glycine residue at a position corresponding to residue 187 in the protein sequence of human ZWINT in the presence of cells expressing a MHC class I molecule of the HLA-B*4403 allele loaded with the above-mentioned peptide (II), wherein X 5 is R in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (ii) if said subject is the subject of (b), culturing CD8 T lymphocytes from a second subject comprising an A at a position corresponding to nucleotide 596 in the nucleic acid sequence of human ZWINT and/or a ZWINT polypeptide comprising an
  • the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising: (a) determining whether a candidate donor expresses a ZWINT nucleic acid comprising an A or a G at a position corresponding to nucleotide 596 in the nucleic acid sequence of human ZWINT ( FIG. 2A , NCBI Reference Sequence: NM_007057.3), and/or a ZWINT polypeptide comprising a arginine or glycine residue at a position corresponding to residue 187 in the protein sequence of human ZWINT ( FIG.
  • the present invention provides a peptide of 50 amino acids or less comprising the sequence (III)
  • Z 1 -X 8 LFFRKVX 9 X 10 -Z 2 (III) wherein Z 1 is an amino terminal modifying group or is absent; X 8 is a sequence of 1 to 43 amino acids or is absent; X 9 is P or A; X 10 is a sequence of 1 to 43 amino acids or is absent; and Z 2 is a carboxy terminal modifying group or is absent.
  • X 8 is serine (S).
  • X 10 is an amino acid, in a further embodiment an aromatic amino acid, more particularly phenylalanine (F).
  • X 9 is P.
  • X 9 is A.
  • the peptide comprises the sequence SLFFRKVPF (SEQ ID NO:19).
  • the peptide is SLFFRKVPF (SEQ ID NO:19). In another embodiment, the peptide comprises the sequence SLFFRKVAF (SEQ ID NO:20). In a further embodiment, the peptide is SLFFRKVAF (SEQ ID NO:20).
  • the present invention provides a method of treating cancer, said method comprising administering to a subject in need thereof an effective amount of CD8 T lymphocytes recognizing a MHC class I molecule of the HLA-B*0801 allele loaded with the above-mentioned peptide.
  • the above-mentioned method further comprises determining whether said subject expresses a MTCH2 nucleic acid comprising a C or a G at a position corresponding to nucleotide 1057 in the nucleic acid sequence of human MTCH2 ( FIG. 3A , NCBI Reference Sequence: NM_014342.3), and/or a MTCH2 polypeptide comprising a proline or alanine residue at a position corresponding to residue 290 in the protein sequence of human MTCH2 ( FIG.
  • the above-mentioned determining comprises sequencing a human MTCH2 nucleic acid.
  • the above-mentioned CD8 T lymphocytes are in vitro expanded CD8 T lymphocytes.
  • the above-mentioned method further comprises: (i) if said subject is the subject of (a), culturing CD8 T lymphocytes from a second subject comprising a G at a position corresponding to nucleotide 1057 in the nucleic acid sequence of human MTCH2 and/or a MTCH2 polypeptide comprising an alanine residue at a position corresponding to residue 290 in the protein sequence of human MTCH2 in the presence of cells expressing a MHC class I molecule of the HLA-B*0801 allele loaded with the above-mentioned peptide (III), wherein X 9 is P in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (ii) if said subject is the subject of (b), culturing CD8 T lymphocytes from a second subject comprising a C at a position corresponding to nucleotide 1057 in the nucleic acid sequence of human MTCH2 and/or a MTCH2 polypeptid
  • the above-mentioned subject is an allogeneic stem cell transplantation (ASCT) recipient.
  • ASCT allogeneic stem cell transplantation
  • the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising: (a) determining whether a candidate donor expresses a MTCH2 nucleic acid comprising a C or a G at a position corresponding to nucleotide 1057 in the nucleic acid sequence of human MTCH2 ( FIG. 3A , NCBI Reference Sequence: NM_014342.3), and/or a MTCH2 polypeptide comprising a proline or alanine residue at a position corresponding to residue 290 in the protein sequence of human MTCH2 ( FIG.
  • the present invention provides a peptide of 50 amino acids or less comprising the sequence (IV)
  • Z 1 -X 11 X 12 VLKPGNX 13 X 14 -Z 2 (IV) wherein Z 1 is an amino terminal modifying group or is absent; X 11 is a sequence of 1 to 43 amino acids or is absent; X 12 is S or T; X 13 is an amino acid or is absent; X 14 is a sequence of 1 to 43 amino acids or is absent; and Z 2 is a carboxy terminal modifying group or is absent.
  • X 11 is absent.
  • the above-mentioned X 13 is an amino acid, in a further embodiment serine (S).
  • X 14 is an amino acid, in a further embodiment a basic amino acid, more particularly lysine (K).
  • X 12 is S.
  • X 12 is T.
  • the above-mentioned peptide comprises the sequence SVLKPGNSK (SEQ ID NO:21). In a further embodiment, the peptide is SVLKPGNSK (SEQ ID NO:21). In another embodiment, the above-mentioned peptide comprises the sequence TVLKPGNSK (SEQ ID NO:22). In a further embodiment, the peptide is TVLKPGNSK (SEQ ID NO:22).
  • the present invention provides a method of treating cancer, said method comprising administering to a subject in need thereof an effective amount of CD8 T lymphocytes recognizing a MHC class I molecule of the HLA-A*0301 allele loaded with the above-mentioned peptide (IV).
  • the above-mentioned method further comprises determining whether said subject expresses an ELF1 nucleic acid comprising an A or T at a position corresponding to nucleotide 1400 in the nucleic acid sequence of human ELF1 ( FIGS. 4A and 4B , NCBI Reference Sequence: NM_172373.3) and/or an ELF1 polypeptide having a threonine or a serine at a position corresponding to residue 343 in the ELF1 protein sequence ( FIG.
  • the above-mentioned determining comprises sequencing a human ELF1 nucleic acid.
  • the above-mentioned CD8 T lymphocytes are in vitro expanded CD8 T lymphocytes.
  • the above-mentioned method further comprises: (i) if said subject is the subject of (a), culturing CD8 T lymphocytes from a second subject comprising a T at a position corresponding to nucleotide 1400 in the nucleic acid sequence of human ELF1 and/or an ELF1 polypeptide comprising a serine residue at a position corresponding to residue 343 in the protein sequence of human ELF1 in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the peptide, wherein X 12 is T in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (ii) if said subject is the subject of (b), culturing CD8 T lymphocytes from a second subject comprising an A at a position corresponding to nucleotide 1400 in the nucleic acid sequence of human ELF1 and/or an ELF1 polypeptide comprising a threonine residue at a position corresponding
  • the above-mentioned subject is an allogeneic stem cell transplantation (ASCT) recipient.
  • ASCT allogeneic stem cell transplantation
  • the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising: (a) determining whether a candidate donor expresses an ELF1 nucleic acid comprising an A or T at a position corresponding to nucleotide 1400 in the nucleic acid sequence of human ELF1 ( FIGS. 4A and 4B , NCBI Reference Sequence: NM_172373.3) and/or an ELF1 polypeptide having a threonine or a serine at a position corresponding to residue 343 in the ELF1 protein sequence ( FIG.
  • the present invention provides a peptide of 50 amino acids or less comprising the sequence V
  • the above-mentioned X 16 is an amino acid, in a further embodiment a methionine (M).
  • X 15 is an amino acid, in a further embodiment an alanine (A).
  • X 18 is an amino acid, in a further embodiment a serine (S).
  • X 19 is an amino acid, in a further embodiment a basic amino acid, more particularly lysine (K).
  • X 17 is R.
  • X 17 is W.
  • the above-mentioned peptide comprises the sequence AMYDKGPFRSK (SEQ ID NO:23).
  • the above-mentioned peptide is AMYDKGPFRSK (SEQ ID NO:23).
  • the above-mentioned peptide comprises the sequence AMYDKGPFWSK (SEQ ID NO:24).
  • the above-mentioned peptide is AMYDKGPFWSK (SEQ ID NO:24).
  • the present invention provides a method of treating cancer, said method comprising administering to a subject in need thereof an effective amount of CD8 T lymphocytes recognizing a MHC class I molecule of the HLA-A*0301 allele loaded with the above-mentioned peptide (V).
  • the above-mentioned method further comprises determining whether said subject expresses an NQO1 nucleic acid comprising an C or T at a position corresponding to nucleotide 615 in the nucleic acid sequence of human NQO1 ( FIG. 5A , NCBI Reference Sequence: NM_000903.2) and/or an NQO1 polypeptide having an arginine or a tryptophan at a position corresponding to residue 139 in the NQO1 protein sequence ( FIG.
  • NCBI Reference Sequence: NP_000894.1 wherein (a) if said subject expresses a NQO1 nucleic acid comprising an C at a position corresponding to nucleotide 615 in the nucleic acid sequence of human NQO1 and/or a NQO1 polypeptide comprising an arginine residue at a position corresponding to residue 139 in the protein sequence of human NQO1, X 17 is R in said peptide; (b) if said subject expresses an NQO1 nucleic acid comprising a T at a position corresponding to nucleotide 615 in the nucleic acid sequence of human NQO1 and/or a NQO1 polypeptide comprising a tryptophan residue at a position corresponding to residue 139 in the protein sequence of human NQO1, X 17 is W in said peptide.
  • the above-mentioned determining comprises sequencing a human NQO1 nucleic acid.
  • the above-mentioned CD8 T lymphocytes are in vitro expanded CD8 T lymphocytes.
  • the above-mentioned method further comprises: (i) if said subject is the subject of (a), culturing CD8 T lymphocytes from a second subject comprising a T at a position corresponding to nucleotide 615 in the nucleic acid sequence of human NQO1 and/or a NQO1 polypeptide comprising a tryptophan residue at a position corresponding to residue 139 in the protein sequence of human NQO1 in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the above-mentioned peptide (V), wherein X 17 is R in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (ii) if said subject is the subject of (b), culturing CD8 T lymphocytes from a second subject comprising a C at a position corresponding to nucleotide 615 in the nucleic acid sequence of human NQO1 and/or a NQO1 poly
  • the above-mentioned subject is an allogeneic stem cell transplantation (ASCT) recipient.
  • ASCT allogeneic stem cell transplantation
  • the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising: (a) determining whether said subject expresses an NQO1 nucleic acid comprising an C or T at a position corresponding to nucleotide 615 in the nucleic acid sequence of human NQO1 ( FIG. 5A , NCBI Reference Sequence: NM_000903.2) and/or an NQO1 polypeptide having an arginine or a tryptophan at a position corresponding to residue 139 in the NQO1 protein sequence ( FIG.
  • the present invention provides a peptide of 50 amino acids or less comprising the sequence VI
  • Z 1 -X 20 X 21 X 22 SLPTSPX 23 X 24 -Z 2 (VI) wherein Z 1 is an amino terminal modifying group or is absent; X 20 is a sequence of 1 to 43 amino acids or is absent; X 21 is an amino acid or is absent; X 22 is an amino acid or is absent; X 23 is G or R; X 24 is a sequence of 1 to 43 amino acids or is absent; Z 2 is a carboxy terminal modifying group or is absent.
  • the peptide has a length of 8 to 12 amino acids.
  • X 22 is an amino acid, in a further embodiment valine (V).
  • X 21 is an amino acid, in a further embodiment arginine (R).
  • X 23 is glycine (G), in another embodiment X 23 is arginine (R).
  • the above-mentioned peptide comprises the sequence RVSLPTSPG (SEQ ID NO:25). In an embodiment, the above-mentioned peptide is RVSLPTSPG (SEQ ID NO:25). In another embodiment, the above-mentioned peptide comprises the sequence RVSLPTSPR (SEQ ID NO:26). In an embodiment, the above-mentioned peptide is RVSLPTSPR (SEQ ID NO:26).
  • the present invention provides a method of treating cancer, said method comprising administering to a subject in need thereof an effective amount of CD8 T lymphocytes recognizing a MHC class I molecule of the HLA-A*0301 allele loaded with the above-mentioned peptide of sequence VI.
  • the above-mentioned method further comprising determining whether said subject expresses a KIAA0226L nucleic acid comprising a G or A at a position corresponding to nucleotide 1059 in the nucleic acid sequence of human KIAA0226L ( FIGS. 6A and 6B , NCBI Reference Sequence: NM_025113.2) and/or an KIAA0226L polypeptide having a glycine or an arginine at a position corresponding to residue 152 in the KIAA0226L protein sequence ( FIG.
  • the determining comprises sequencing a human KIAA0226L nucleic acid.
  • the above-mentioned CD8 T lymphocytes are in vitro expanded CD8 T lymphocytes.
  • the above-mentioned method further comprises: (i) if said subject is the subject of (a) above, culturing CD8 T lymphocytes from a second subject comprising an A at a position corresponding to nucleotide to nucleotide 1059 in the nucleic acid sequence of human KIAA0226L and/or a KIAA0226L polypeptide comprising an arginine residue at a position corresponding to residue 152 in the protein sequence of human KIAA0226L in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the peptide of sequence VI, wherein X 23 is G in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (ii) if said subject is the subject of (b) above, culturing CD8 T lymphocytes from a second subject comprising a G at a position corresponding to nucleotide 1059 in the nucleic acid sequence of human KIAA0226
  • the above-mentioned subject is an allogeneic stem cell transplantation (ASCT) recipient.
  • ASCT allogeneic stem cell transplantation
  • the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising: (a) determining whether a candidate donor expresses a KIAA0226L nucleic acid comprising a G or A at a position corresponding to nucleotide 1059 in the nucleic acid sequence of human KIAA0226L ( FIGS. 6A and 6B , NCBI Reference Sequence: NM_025113.2) and/or an KIAA0226L polypeptide having a glycine or an arginine at a position corresponding to residue 152 in the KIAA0226L protein sequence ( FIG.
  • the present invention provides a peptide of 50 amino acids or less comprising the sequence VII
  • Z 1 is an amino terminal modifying group or is absent;
  • X 25 is a sequence of 1 to 43 amino acids or is absent;
  • X 26 is an amino acid or is absent;
  • X 27 is an amino acid or is absent;
  • X 28 is K or N;
  • X 29 is a sequence of 1 to 43 amino acids or is absent;
  • Z 2 is a carboxy terminal modifying group or is absent.
  • the peptide has a length of 8 to 12 amino acids.
  • X 27 is an amino acid, more specifically methionine (M).
  • X 26 is an amino acid, more specifically valine (V).
  • X 28 is K.
  • X 28 is N.
  • the peptide comprises the sequence VMGNPGTFK (SEQ ID NO: 27). In a further embodiment, the peptide is VMGNPGTFK (SEQ ID NO: 27). In another embodiment, the peptide comprises the sequence VMGNPGTFN (SEQ ID NO: 28). In an embodiment, the peptide is VMGNPGTFN (SEQ ID NO: 28).
  • the present invention provides a method of treating cancer, said method comprising administering to a subject in need thereof an effective amount of CD8 T lymphocytes recognizing a MHC class I molecule of the HLA-A*0301 allele loaded with the above-mentioned peptide of sequence VII.
  • the above-mentioned method further comprises determining whether said subject expresses an RMDN1 nucleic acid comprising an A or C at a position corresponding to nucleotide 316 in the nucleic acid sequence of human RMDN1 ( FIG. 7A , NCBI Reference Sequence: NM_016033.2) and/or an RMDN1 polypeptide having a lysine or an asparagine at a position corresponding to residue 52 in the RMDN1 protein sequence ( FIG.
  • the determining comprises sequencing a human RMDN1 nucleic acid.
  • the above-mentioned CD8 T lymphocytes are in vitro expanded CD8 T lymphocytes.
  • the above-mentioned method further comprises: (i) if said subject is the subject of (a), culturing CD8 T lymphocytes from a second subject comprising a C at a position corresponding to nucleotide 316 in the nucleic acid sequence of human RMDN1 and/or a RMDN1 polypeptide comprising an asparagine residue at a position corresponding to residue 52 in the protein sequence of human RMDN1 in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the peptide of sequence VII, wherein X 28 is K in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (ii) if said subject is the subject of (b) above, culturing CD8 T lymphocytes from a second subject comprising an A at a position corresponding to nucleotide 316 in the nucleic acid sequence of human RMDN1 and/or a RMDN1 polypeptide comprising a
  • the above-mentioned subject is an allogeneic stem cell transplantation (ASCT) recipient.
  • ASCT allogeneic stem cell transplantation
  • the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising: (a) determining whether a candidate donor expresses an RMDN1 nucleic acid comprising an A or C at a position corresponding to nucleotide 316 in the nucleic acid sequence of human RMDN1 ( FIG. 7A , NCBI Reference Sequence: NM_016033.2) and/or an RMDN1 polypeptide having a lysine or an asparagine at a position corresponding to residue 52 in the RMDN1 protein sequence ( FIG.
  • Z 1 is absent in the above-mentioned peptide (I)-(VII).
  • Z 2 is absent in the above-mentioned peptide (I)-(VII).
  • the present invention provides a MiHA identified by the above-mentioned method.
  • the MiHA is a peptide of sequences (I)-(VII) as defined herein.
  • the present invention provides a nucleic acid encoding the above-mentioned peptide (I)-(VII).
  • the present invention provides an isolated major histocompatibility complex (MHC) class I molecule loaded with the peptide (I)-(VII).
  • MHC major histocompatibility complex
  • the present invention provides an isolated cell expressing at its surface a MHC class I molecule loaded with the above-mentioned peptide (I)-(VII).
  • the major histocompatibility complex (MHC) class I molecule is of the HLA-A*0301, HLA-B*0801 or HLA-B*4403 allele. In a further embodiment, the major histocompatibility complex (MHC) class I molecule is of the HLA-A*0301 allele. In another embodiment, the major histocompatibility complex (MHC) class I molecule is of the HLA-B*0801 allele. In another embodiment, the major histocompatibility complex (MHC) class I molecule is of the HLA-B*4403 allele.
  • FIGS. 1A to C show the nucleotide sequence of human centromer protein F, 350/400 kDa (mitosin) (CENPF) cDNA (SEQ ID NO:1). The coding region is in italics;
  • FIG. 1D shows the amino acid sequence of human CENPF polypeptide (SEQ ID NO:2)
  • FIG. 2A shows the nucleotide sequence of human ZW10 interactor (ZWINT) cDNA (SEQ ID NO:3).
  • the coding region is in italics;
  • FIG. 2B shows the amino acid sequence of human ZWINT polypeptide (SEQ ID NO:4)
  • FIG. 3A shows the nucleotide sequence of human mitochondrial carrier homolog 2 (MTCH2) cDNA (SEQ ID NO:5). The coding region is in italics;
  • FIG. 3B shows the amino acid sequence of human MTCH2 polypeptide (SEQ ID NO:6)
  • FIGS. 4A and 4B show the nucleotide sequence of human ELF1 [E74-like factor 1 (ets domain transcription factor)] cDNA (SEQ ID NO:7).
  • the coding region is in italics;
  • FIG. 4C shows the amino acid sequence of human ELF1 polypeptide (SEQ ID NO:8)
  • FIGS. 5A and 5B show the nucleotide sequence of human NQO1 [NAD(P)H dehydrogenase, quinone 1] cDNA (SEQ ID NO:9).
  • the coding region is in italics;
  • FIG. 5C shows the amino acid sequence of human NQO1 polypeptide (SEQ ID NO:10)
  • FIGS. 6A and 6B show the nucleotide sequence of human KIAA0226L cDNA (SEQ ID NO:11). The coding region is in italics;
  • FIG. 6C shows the amino acid sequence of human KIAA0226L polypeptide (SEQ ID NO:12);
  • FIG. 7A shows the nucleotide sequence of human RMDN1 cDNA (SEQ ID NO:13). The coding region is in italics; and
  • FIG. 7B shows the amino acid sequence of human RMDN1 polypeptide (SEQ ID NO:14).
  • Described herein is a novel method for human MiHA discovery, novel MiHAs identified using this method, as well as uses of the novel MiHAs.
  • One of the features of the method is the inclusion of personalized translated transcriptome and/or exome in the database used for peptide identification by mass spectroscopy (MS).
  • MS mass spectroscopy
  • Candidate MiHAs are identified by comparing the personalized transcriptome and/or exome to a reference genome and/or to the transcriptome and/or exome of an HLA-matched subject (e.g., an HLA-identical sibling).
  • the present invention provides a method of identifying a minor histocompatibility antigen (MiHA) candidate, the method comprising: (a) isolating and determining the sequence of MHC-associated peptides (MAPs) in a first cell sample from a first subject; (b) performing a whole transcriptome and/or exome sequencing on a second cell sample obtained from said first subject; (c) comparing the sequenced whole transcriptome and/or exome to a reference genome to identify single nucleotide variations (SNVs) between the transcriptome and/or exome of said first subject and the reference genome; (d) in silico translating the sequences containing the identified SNVs to identify peptide sequences comprising at least one non-synonymous mutation caused by said SNVs; (e) comparing the sequences of the MAPs isolated in (a) with the peptide sequences identified in (d); and (f) identifying a MiHA candidate based on said comparison.
  • MiHA minor histocompatibility antigen
  • the MiHA candidate is a MAP whose sequence comprises at least one mutation relative to the corresponding sequence translated from the reference genome.
  • the present invention provides a method of identifying a minor histocompatibility antigen (MiHA) candidate, the method comprising: (a) isolating MHC-associated peptides (MAPs) in a first cell sample from a first and second subjects, wherein said first and second subjects are human leukocyte antigen (HLA)-matched; (b) performing a whole transcriptome and/or exome sequencing on a second cell sample obtained from said first and second subjects; (c) comparing the sequenced whole transcriptomes and/or exomes to identify single nucleotide variations (SNVs) between the transcriptomes and/or exomes of said first and second subjects; (d) in silico translating the sequences containing the identified SNVs to identify peptide sequences comprising at least one non-synonymous mutation caused by said SNVs; (e) comparing the sequences of the MAPs isolated in (a) with the peptide sequences identified in (d); and (f) identifying a MiHA candidate
  • reference genome refers to the human genome assemblies reported in the literature, and includes for example the Genome Reference Consortium Human Build 37 (GRCh37, Genome Reference Consortium; The International Human Genome Sequencing Consortium. Nature. 2004; 431:931-945), Hs_Celera_WGSA (Celera Genomics; Istrail S. et al., Proc Natl Acad Sci USA. 2004 Feb. 17; 101(7):1916-21). Epub 2004 Feb. 9), HuRef and HuRefPrime (J. Craig Venter Institute; Levy S, et al. PLoS Biology. 2007; 5:2113-2144), YH1 and BGIAF (Beijing Genomics Institute; Li R, et al. Genome Research. 2010; 20: 265-272), as well as HsapALLPATHS1 (Broad Institute).
  • the reference genome is GRCh37.
  • the above-noted first sample may be from any source that contains cells expressing MHC class I molecules, including a tissue or body fluid from the subject, such as blood, serum, immune cells (e.g., lymphocytes), blood cells (e.g., PBMCs or a subset thereof), tissues, or a cell line derived from primary cells.
  • the first sample is a blood cell sample, for example a PBMC sample, or a cell line derived from blood cells such as PBMCs (e.g., an immortalized cell line).
  • Methods for generating a cell line from primary cells, or for immortalizing primary cells include, for example, immortalization of primary cells by recombinant expression of human telomerase reverse transcriptase (TERT) (Barsov E V, Curr Protoc Immunol. 2011 November; Chapter 7: Unit 7.21B), immortalization by recombinant expression of viral genes such as Simian virus 40 (SV40) T antigen, adenovirus E1A and E1B, human papillomavirus (HPV) E6 and E7 and Epstein-Barr Virus (EBV), as well as inactivation of tumor suppression genes such as p53 or Rb.
  • SV40 Simian virus 40
  • HPV human papillomavirus
  • E6 and E7 Epstein-Barr Virus
  • the first sample is an immortalized cell line derived from primary cells obtained from the subject, in a further embodiment an immortalized B cell line, such as an EBV-transformed B lymphoblastoid cell line (B-LCL).
  • B-LCL EBV-transformed B lymphoblastoid cell line
  • MHC-associated peptides Methods for isolating MHC-associated peptides (MAPs) from a cell sample are well known in the art.
  • the most commonly used technique is mild acid elution (MAE) of MHC-associated peptides from living cells, as described in Fortier et al. ( J. Exp. Med. 205(3): 595-610, 2008).
  • Another technique is immunoprecipitation or affinity purification of peptide-MHC class I complexes followed by peptide elution (see, e.g., Gebreselassie et al., Hum Immunol. 2006 November; 67(11): 894-906).
  • Two high-throughput strategies based on the latter approach have been implemented.
  • the first is based on transfection of cell lines with expression vectors coding soluble secreted MHCs (lacking a functional transmembrane domain) and elution of peptides associated with secreted MHCs (Barnea et al., Eur J Immunol. 2002 January; 32(1):213-22; and Hickman H D et al., J Immunol. 2004 Mar. 1; 172(5):2944-52).
  • the second approach hinges on chemical or metabolic labeling to provide quantitative profiles of MHC-associated peptides (Weinzierl A O et al., Mol Cell Proteomics. 2007 January; 6(1):102-13. Epub 2006 Oct. 29; Lemmel C et al., Nat Biotechnol. 2004 April; 22(4):450-4. Epub 2004 Mar. 7; Milner E, Mol Cell Proteomics. 2006 February; 5(2):357-65. Epub 2005 Nov. 4).
  • Eluted MAPs may be subjected to any purification/enrichment steps, including size exclusion chromatography or ultrafiltration (using a filter with a cut-off of about 5000 Da, for example about 3000 Da), and/or ion exchange chromatography (e.g., cation exchange chromatography), prior to further analysis.
  • the sequence of the eluted MAPs may be determined using any method known in the art for sequencing peptides/proteins, such as mass spectroscopy (as described below) and the Edman degradation reaction.
  • the above-noted second sample may be from any source that contains genomic DNA, RNA, and/or proteins, for example a tissue or body fluid from the subject, such as blood, serum, immune cells (e.g., lymphocytes), blood cells (e.g., PBMCs), tissues, or a cell line derived from primary cells (as described above).
  • the second sample is an immortalized cell line derived from primary cells obtained from the subject, in a further embodiment an immortalized B cell line, such as an EBV-transformed B lymphoblastoid cell line (B-LCL).
  • B-LCL EBV-transformed B lymphoblastoid cell line
  • the cell sample may be subjected to commonly used isolation and/or purification techniques for enrichment in nucleic acids (genomic DNA, mRNA) and/or proteins.
  • transcriptome libraries are generated/constructed from the RNA obtained from the sample.
  • Transcriptome library construction may include one or more of the following steps: poly-A mRNA enrichment/purification; RNA fragmentation and priming for cDNA synthesis; reverse transcription (RT) (using random primers); second round of RT to generate a double-stranded cDNA, cDNA purification; end repair of fragmented cDNA, adenylation of the 3′ ends, ligation of adaptors and enrichment of DNA fragments containing adapter molecules.
  • Kits suitable for transcriptome library construction are commercially available, for example from Life Technologies (Ambion® RNA-Seq Library Construction Kit), Applied Biosystems (AB Library BuilderTM Whole Transcriptome Core Kit), Qiagen (QuantilectTM Whole Transcriptome Kit) and Sigma-Aldrich (TransPlex® Complete Whole Transcriptome Amplification Kit)
  • genomic libraries are generated/constructed from the genomic DNA obtained from the sample.
  • Genomic library construction may include one or more of the following steps: DNA shearing, DNA end repair, 3′ ends adenylation, ligation of adaptors, purification of ligation products and amplification (e.g., PCR) to enrich DNA fragments that have adapter molecules.
  • Kits suitable for genomic library construction are commercially available, for example from Illumina (TruSeqTM DNA Sample Preparation Kit (v2) (Cat. No. FC-930-1021), Life Technologies (SOLiD® Fragment Library Construction Kit and New England BioLabs (NEBNext® DNA Library Preparation).
  • the genomic (DNA-Seq) libraries are subjected to an enrichment step to sequence only the coding portion (exome) of the human genome.
  • Kits suitable for exome enrichment are commercially available, for example from Illumina (TruSeqTM exome enrichment kit, FC-930-1012), Life Technologies (TargetSeqTM Exome and Custom Enrichment System, A14060-A14063), FlexGen (FleXome whole exome enrichment kit v2), Roche NimbleGen (SeqCap EZ Human Exome Library v2.0) and Agilent Technologies (SureSelect All Exon kits)
  • RNA-Seq Methods to perform whole transcriptome or exome sequencing (RNA-Seq) are known in the art (see, for example, Wang et al., Nature Reviews Genetics 10, 57-63, January 2009; Genome Biology 2011, 12(9), Exome sequencing special issue).
  • SNVs single nucleotide variations
  • SNPs single nucleotide polymorphisms
  • the in silico translation of nucleic acid sequences to protein sequences may be performed using any suitable softwares or tools, including the ExPASy Translate tool, Vector NTITM (Life Technologies), pyGeno (Granados et al., 2012), Virtual Ribosome (CBS, University of Denmark), etc.
  • the in silico translation of transcriptomes and/or exomes permits the identification of peptide sequences comprising at least one non-synonymous mutation caused by the SNVs.
  • all possible amino acid (aa) sequence variants of 15 amino acids or less (in embodiments, 14, 13, 12, 11 amino acids or less) comprising at least one non-synonymous mutation are computed, listed and used in the comparing step (e).
  • a window of 90 bp (84, 78, 72 or 66 bp) around each one of the polymorphic positions is computed to obtain the list of every possible amino acid (aa) sequence variant defined by these 90 bp (84, 78, 72 or 66 bp) (30, 28, 26, 24 or 22 aa) windows.
  • a list of most possible aa sequences of at most 15 aa (in embodiments, 14, 13, 12, 11 aa) affected by non-synonymous polymorphisms may be obtained.
  • all possible amino acid (aa) sequence variants of for example 30 amino acids or less (in embodiments, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12 amino acids or less) comprising at least one non-synonymous mutation are computed, listed and used in the comparing step (e).
  • a window of 180 bp around each one of the polymorphic positions is computed to obtain the list of every possible amino acid (aa) sequence variant defined by these 180 bp (60 aa) windows. In this way, a list of most possible aa sequences of at most 30 aa affected by non-synonymous polymorphisms may be obtained.
  • all possible amino acid (aa) sequence variants of 12 or 11 amino acids or less comprising at least one non-synonymous mutation are computed, listed and used in the comparing step (e).
  • a window of 72 or 66 bp around each one of the polymorphic positions is computed to obtain the list of every possible amino acid (aa) sequence variant defined by these 72 or 66 bp (24 or 22 aa) windows.
  • a list of most possible aa sequences of at most 12 or 11 aa affected by non-synonymous polymorphisms was obtained.
  • the above-noted peptide sequences have a length of about 7 to about 15 amino acids (e.g., 7, 8, 9, 10, 11, 12, 13, 14 or 15), in a further embodiment of about 8 or 9 to about 11 or 12 amino acids (e.g., 8, 9, 10, 11 or 12).
  • the comparison of the sequences of the MAPs isolated from the first sample with the peptide sequences derived from the transcriptome and/or exome identified above comprises subjecting the isolated MAPs to mass spectrometry and comparing the MS spectra obtained with the peptide sequences derived from the transcriptome and/or exome.
  • the mass spectrometry is liquid chromatography-mass spectrometry (LC-MS), in a further embodiment LC-MS coupled to peptide mass fingerprinting (LC-MS/MS).
  • the method further comprises determining the binding of the MiHA candidate identified to a MHC class I molecule.
  • the binding may be a predicted binding affinity (IC 50 ) of peptides to the allelic products, which may be obtained using tools such as the NetMHCcons software version 1.0 (http://www.cbs.dtu.dk/services/NetMHCcons/) (Karosiene et al., 2011).
  • NetMHCcons software version 1.0 http://www.cbs.dtu.dk/services/NetMHCcons/
  • An overview of the various available MHC class I peptide binding tools is provided in Peters B et al., PLoS Comput Biol 2006, 2(6):e65; Trost et al., Immunome Res 2007, 3(1):5; Lin et al., BMC Immunology 2008, 9:8)
  • peptides with a predicted IC 50 below 50 nM are considered as strong binders and peptides with an IC 50 between about 50 and about 500 nM are considered as weak binders.
  • the binding of the MiHA candidate identified to a MHC class I molecule may be determined using other known methods, for example the 12 Peptide Binding Assay.
  • 12 cell lines are deficient in TAP but still express low amounts of MHC class I on the surface of the cells.
  • the T2 binding assay is based upon the ability of peptides to stabilize the MHC class I complex on the surface of the T2 cell line.
  • T2 cells are incubated with a specific peptide (e.g., a candidate MiHA), stabilized MHC class I complexes are detected using a pan-HLA class I antibody, an analysis is carried out (by flow cytometry, for example) and binding is assessed in relation to a non-binding negative control.
  • the presence of stabilized peptide/MHC class I complexes at the surface is indicative that the peptide (e.g., candidate MiHA) binds to MHC class I molecules.
  • the binding of a peptide of interest (e.g., candidate MiHA) to MHC may also be assessed based on its ability to inhibit the binding of a radiolabeled probe peptide to MHC molecules.
  • MHC molecules are solubilized with detergents and purified by affinity chromatography. They are then incubated for 2 days at room temperature with the inhibitor peptide (e.g., candidate MiHA) and an excess of a radiolabeled probe peptide, in the presence of a cocktail of protease inhibitors. At the end of the incubation period, MHC-peptide complexes are separated from unbound radiolabeled peptide by size-exclusion gel-filtration chromatography, and the percent bound radioactivity is determined.
  • the binding affinity of a particular peptide for an MHC molecule may be determined by co-incubation of various doses of unlabeled competitor peptide with the MHC molecules and labeled probe peptide.
  • concentration of unlabeled peptide required to inhibit the binding of the labeled peptide by 50% (IC50) can be determined by plotting dose versus % inhibition (see, e.g., Current Protocols in Immunology (1998) 18.3.1-18.3.19, John Wiley & Sons, Inc.).
  • the binding of the MiHA candidate identified to a MHC class I molecule may be determined using an epitope discovery system, such as the ProImmune REVEAL & ProVE® epitope discovery system.
  • the present invention provides a peptide (e.g., an isolated or synthetic peptide) of 50 amino acids or less comprising the sequence (I) to (VII) described herein.
  • a peptide e.g., an isolated or synthetic peptide
  • the present invention provides a peptide (e.g., an isolated or synthetic peptide) of 50 amino acids or less comprising the sequence (I):
  • Z1 is an amino terminal modifying group or is absent;
  • X1 is a sequence of 1 to 43 amino acids or is absent;
  • X2 is L or S;
  • X3 is a sequence of 1 to 43 amino acids or is absent; and
  • Z2 is a carboxy terminal modifying group or is absent.
  • the present invention provides a peptide (e.g., an isolated or synthetic peptide) of 50 amino acids or less comprising the sequence (II):
  • Z 1 is an amino terminal modifying group or is absent;
  • X 4 is a sequence of 1 to 43 amino acids or is absent;
  • X 5 is G or R;
  • X 6 is an amino acid or is absent;
  • X 7 is a sequence of 1 to 43 amino acids or is absent; and
  • Z 2 is a carboxy terminal modifying group or is absent.
  • the present invention provides a peptide (e.g., an isolated or synthetic peptide) of 50 amino acids or less comprising the sequence (III):
  • Z 1 is an amino terminal modifying group or is absent;
  • X 8 is a sequence of 1 to 43 amino acids or is absent;
  • X 9 is P or A;
  • X 10 is a sequence of 1 to 43 amino acids or is absent; and
  • Z 2 is a carboxy terminal modifying group or is absent.
  • the present invention provides a peptide (e.g., an isolated or synthetic peptide) of 50 amino acids or less comprising the sequence (IV):
  • Z 1 is an amino terminal modifying group or is absent;
  • X 11 is a sequence of 1 to 43 amino acids or is absent;
  • X 12 is S or T;
  • X 13 is an amino acid or is absent;
  • X 14 is a sequence of 1 to 43 amino acids or is absent; and
  • Z 2 is a carboxy terminal modifying group or is absent.
  • the present invention provides a peptide (e.g., an isolated or synthetic peptide) of 50 amino acids or less comprising the sequence (V):
  • Z 1 is an amino terminal modifying group or is absent;
  • X 15 is a sequence of 1 to 43 amino acids or is absent;
  • X 16 is an amino acid or is absent;
  • X 17 is R or W;
  • X 18 is an amino acid or is absent;
  • X 19 is a sequence of 1 to 43 amino acids or is absent;
  • Z 2 is a carboxy terminal modifying group or is absent.
  • the present invention provides a peptide (e.g., an isolated or synthetic peptide) of 50 amino acids or less comprising the sequence (VI):
  • Z 1 is an amino terminal modifying group or is absent;
  • X 20 is a sequence of 1 to 43 amino acids or is absent;
  • X 21 is an amino acid or is absent;
  • X 22 is an amino acid or is absent;
  • X 23 is G or R;
  • X 24 is a sequence of 1 to 43 amino acids or is absent;
  • Z 2 is a carboxy terminal modifying group or is absent.
  • the present invention provides a peptide (e.g., an isolated or synthetic peptide) of 50 amino acids or less comprising the sequence (VII):
  • Z 1 is an amino terminal modifying group or is absent
  • X 25 is a sequence of 1 to 43 amino acids or is absent
  • X 26 is an amino acid or is absent
  • X 27 is an amino acid or is absent
  • X 28 is K or N
  • X 29 is a sequence of 1 to 43 amino acids or is absent
  • Z 2 is a carboxy terminal modifying group or is absent.
  • the present invention provides a peptide of 50 amino acids or less comprising any one of the sequences (I) to (VII) as defined herein.
  • peptides presented in the context of HLA class I vary in length from about 7 to about 15 amino acid residues, and a longer peptide (e.g., of 50 amino acids or less) can be enzymatically processed to a peptide of such length.
  • the peptide is 45, 40, 35, 30, 25, 20 or 15 amino acids or less.
  • a peptide comprising the above-noted sequence/motif provided by the invention typically is at least 7 amino acids in length but preferably at least 8 or 9 amino acids.
  • the upper length of a peptide provided by the invention is no more than 15 amino acids, but preferably no more than about 13, 12 or 11 amino acids in length.
  • the above-mentioned peptide is about 8 to 12 amino acids long (e.g., 8, 9, 10, 11 or 12 amino acids long), small enough for a direct fit in an HLA class I molecule, but it may also be larger, between 12 to about 20, 25, 30, 35, 40, 45 or 50 amino acids and presented by HLA molecules only after cellular uptake and intracellular processing by the proteasome and transport before presentation in the groove of an MHC molecule.
  • amino acid as used herein includes both L- and D-isomers of the naturally occurring amino acids as well as other amino acids (e.g., naturally-occurring amino acids, non-naturally-occurring amino acids, amino acids which are not encoded by nucleic acid sequences, etc.) used in peptide chemistry to prepare synthetic analogs of peptides.
  • naturally occurring amino acids are glycine, alanine, valine, leucine, isoleucine, serine, threonine, etc.
  • Non-genetically encoded forms of amino acids include for example non-genetically encoded forms of amino acids, as well as a conservative substitution of an L-amino acid.
  • Naturally-occurring non-genetically encoded amino acids include, for example, beta-alanine, 3-amino-propionic acid, 2,3-diaminopropionic acid, alpha-aminoisobutyric acid (Aib), 4-amino-butyric acid, N-methylglycine (sarcosine), hydroxyproline, ornithine (e.g., L-ornithine), citrulline, t-butylalanine, t-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine, norleucine (Nle), norvaline, 2-naphthylalanine, pyridylalanine, 3-benzothienyl alanine, 4-chloropheny
  • the peptides of the present invention include peptides with altered sequences containing substitutions of functionally equivalent amino acid residues, relative to the above-mentioned sequences.
  • one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity (having similar physico-chemical properties) which acts as a functional equivalent, resulting in a silent alteration.
  • Substitution for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • positively charged (basic) amino acids include arginine, lysine and histidine (as well as homoarginine and ornithine).
  • Nonpolar (hydrophobic) amino acids include leucine, isoleucine, alanine, phenylalanine, valine, proline, tryptophan and methionine.
  • Uncharged polar amino acids include serine, threonine, cysteine, tyrosine, asparagine and glutamine.
  • Negatively charged (acidic) amino acids include glutamic acid and aspartic acid.
  • the amino acid glycine may be included in either the nonpolar amino acid family or the uncharged (neutral) polar amino acid family. Substitutions made within a family of amino acids are generally understood to be conservative substitutions.
  • the above-mentioned peptide may comprise all L-amino acids, all D-amino acids or a mixture of L- and D-amino acids. In an embodiment, the above-mentioned peptide comprises all L-amino acids.
  • the peptide may also be N- and/or C-terminally capped or modified to prevent degradation, increase stability or uptake.
  • the amino terminal residue (i.e., the free amino group at the N-terminal end) of the peptide is modified (e.g., for protection against degradation), for example by covalent attachment of a moiety/chemical group (Z 1 ).
  • Z 1 may be a straight chained or branched alkyl group of one to eight carbons, or an acyl group (R—CO—), wherein R is a hydrophobic moiety (e.g., acetyl, propionyl, butanyl, iso-propionyl, or iso-butanyl), or an aroyl group (Ar—CO—), wherein Ar is an aryl group.
  • R—CO— acyl group
  • the acyl group is a C 1 -C 16 or C 3 -C 16 acyl group (linear or branched, saturated or unsaturated), in a further embodiment, a saturated C 1 -C 6 acyl group (linear or branched) or an unsaturated C 3 -C 6 acyl group (linear or branched), for example an acetyl group (CH 3 —CO—, Ac).
  • Z 1 is absent.
  • the carboxy terminal residue (i.e., the free carboxy group at the C-terminal end of the peptide) of the peptide may be modified (e.g., for protection against degradation), for example by amidation (replacement of the OH group by a NH 2 group), thus in such a case Z 2 is a NH 2 group.
  • Z 2 may be an hydroxamate group, a nitrile group, an amide (primary, secondary or tertiary) group, an aliphatic amine of one to ten carbons such as methyl amine, iso-butylamine, iso-valerylamine or cyclohexylamine, an aromatic or arylalkyl amine such as aniline, napthylamine, benzylamine, cinnamylamine, or phenylethylamine, an alcohol or CH 2 OH.
  • Z 2 is absent.
  • the peptide comprises the sequence ELQEKFLSL (SEQ ID NO:15), in a further embodiment the peptide is ELQEKFLSL (SEQ ID NO:15). In another embodiment, the peptide comprises the sequence ELQEKFSSL (SEQ ID NO:16), in a further embodiment the peptide is ELQEKFSSL (SEQ ID NO:16).
  • the peptide comprises the sequence QELDGVFQKL (SEQ ID NO:17). In a further embodiment, the peptide is QELDGVFQKL (SEQ ID NO:17). In another embodiment, the peptide comprises the sequence QELDRVFQKL (SEQ ID NO:18). In a further embodiment, the peptide is QELDRVFQKL (SEQ ID NO:18).
  • the peptide comprises the sequence SLFFRKVPF (SEQ ID NO:19). In a further embodiment, the peptide is SLFFRKVPF (SEQ ID NO:19). In another embodiment, the peptide comprises the sequence SLFFRKVAF (SEQ ID NO:20). In a further embodiment, the peptide is SLFFRKVAF (SEQ ID NO:20).
  • the above-mentioned peptide comprises the sequence SVLKPGNSK (SEQ ID NO:21). In a further embodiment, the peptide is SVLKPGNSK (SEQ ID NO:21). In another embodiment, the above-mentioned peptide comprises the sequence TVLKPGNSK (SEQ ID NO:22). In a further embodiment, the peptide is TVLKPGNSK (SEQ ID NO:22).
  • the above-mentioned peptide comprises the sequence AMYDKGPFRSK (SEQ ID NO:23). In an embodiment, the above-mentioned peptide is AMYDKGPFRSK (SEQ ID NO:23). In another embodiment, the above-mentioned peptide comprises the sequence AMYDKGPFVVSK (SEQ ID NO:24). In an embodiment, the above-mentioned peptide is AMYDKGPFVVSK (SEQ ID NO:24).
  • the above-mentioned peptide comprises the sequence RVSLPTSPG (SEQ ID NO:25). In an embodiment, the above-mentioned peptide is RVSLPTSPG (SEQ ID NO:25). In another embodiment, the above-mentioned peptide comprises the sequence RVSLPTSPR (SEQ ID NO:26). In an embodiment, the above-mentioned peptide is RVSLPTSPR (SEQ ID NO:26).
  • the above-mentioned peptide comprises the sequence VMGNPGTFK (SEQ ID NO:27). In an embodiment, the above-mentioned peptide is VMGNPGTFK (SEQ ID NO:27). In another embodiment, the above-mentioned peptide comprises the sequence VMGNPGTFN (SEQ ID NO:28). In an embodiment, the above-mentioned peptide is VMGNPGTFN (SEQ ID NO:28).
  • the peptides of the invention may be produced by expression in a host cell comprising a nucleic acid encoding the peptides (recombinant expression) or by chemical synthesis (e.g., solid-phase peptide synthesis).
  • Peptides can be readily synthesized by manual and/or automated solid phase procedures well known in the art. Suitable syntheses can be performed for example by utilizing “T-boc” or “Fmoc” procedures. Techniques and procedures for solid phase synthesis are described in for example Solid Phase Peptide Synthesis: A Practical Approach, by E. Atherton and R. C. Sheppard, published by IRL, Oxford University Press, 1989.
  • the peptides may be prepared by way of segment condensation, as described, for example, in Liu et al., Tetrahedron Lett. 37: 933-936, 1996; Baca et al., J. Am. Chem. Soc. 117: 1881-1887, 1995; Tam et al., Int. J. Peptide Protein Res. 45: 209-216, 1995; Schnolzer and Kent, Science 256: 221-225, 1992; Liu and Tam, J. Am. Chem. Soc. 116: 4149-4153, 1994; Liu and Tam, Proc. Natl. Acad. Sci. USA 91: 6584-6588, 1994; and Yamashiro and Li, Int. J. Peptide Protein Res. 31: 322-334, 1988).
  • Other methods useful for synthesizing the peptides are described in Nakagawa et al., J. Am. Chem. Soc. 107: 7087-7092, 1985.
  • Peptides comprising naturally occurring amino acids encoded by the genetic code may also be prepared using recombinant DNA technology using standard methods.
  • the invention further provides a nucleic acid (isolated) encoding the above-mentioned peptides of sequences I-VII.
  • the nucleic acid does not encode the full-length CENPF, ZWINT, MTCH2, ELF1, NQO1, KIAA0226L or RMDN1 polypeptide.
  • the nucleic acid has a length of 150 nucleotides or less, in further embodiments 135, 120, 105, 90, 75, 60, 45, 42 or 39 nucleotides or less.
  • the nucleic acid comprises from about 21 nucleotides to about 45 nucleotides, from about 24 to about 36 nucleotides, for example 24, 27, 30, 33 or 36 nucleotides.
  • isolated refers to a peptide or nucleic molecule separated from other components that are present in the natural source of the macromolecule (other nucleic acids, proteins, lipids, sugars, etc.).
  • synthetic refers to a peptide or nucleic molecule that is not isolated from its natural sources, e.g., which is produced through recombinant technology or using chemical synthesis.
  • the above-mentioned peptide is substantially pure.
  • a compound is “substantially pure” when it is separated from the components that naturally accompany it.
  • a compound is substantially pure when it is at least 60%, more generally 75%, 80% or 85%, preferably over 90% and more preferably over 95%, by weight, of the total material in a sample.
  • a polypeptide that is chemically synthesized or produced by recombinant technology will generally be substantially free from its naturally associated components.
  • a nucleic acid molecule is substantially pure when it is not immediately contiguous with (i.e., covalently linked to) the coding sequences with which it is normally contiguous in the naturally occurring genome of the organism from which the nucleic acid is derived.
  • a substantially pure compound can be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid molecule encoding a peptide compound; or by chemical synthesis. Purity can be measured using any appropriate method such as column chromatography, gel electrophoresis, HPLC, etc.
  • the nucleic acid may be in a vector, such as a cloning vector or an expression vector, that may be transfected into a host cell.
  • the nucleic acid may be incorporated into the genome of the host cell.
  • the host cell expresses the nucleic acid, and in turn the encoded peptide.
  • the invention also provides a vector or plasmid comprising the above-mentioned nucleic acid.
  • the vector or plasmid contains the necessary elements for the transcription and translation of the inserted coding sequence, and may contain other components such as resistance genes, cloning sites, etc.
  • operably linked refers to a juxtaposition of components, particularly nucleotide sequences, such that the normal function of the components can be performed.
  • a coding sequence that is operably linked to regulatory sequences refers to a configuration of nucleotide sequences wherein the coding sequences can be expressed under the regulatory control, that is, transcriptional and/or translational control, of the regulatory sequences.
  • regulatory/control region or “regulatory/control sequence”, as used herein, refers to the non-coding nucleotide sequences that are involved in the regulation of the expression of a coding nucleic acid.
  • regulatory region includes promoter sequences, regulatory protein binding sites, upstream activator sequences, and the like.
  • the present invention provides an MHC class I molecule loaded with the above-mentioned peptide.
  • the MHC molecule is a HLA-B8 molecule, in a further embodiment a HLA-B*0801 molecule.
  • the peptide is non-covalently bound to the MHC class I molecule (i.e., the peptide is loaded into the peptide binding groove/pocket but is not covalently attached to the MHC class I molecule).
  • the peptide is covalently attached/bound to the MHC class I molecule.
  • the peptide and the MHC class I molecule are produced as a fusion protein, typically with a short (e.g., 5 to 20 residues, preferably about 10) flexible linker or spacer (e.g., a polyglycine linker).
  • the invention provides a nucleic acid encoding a peptide-MHC class I fusion protein.
  • the MHC class I molecule-peptide complex is multimerized. Accordingly, in another aspect, the present invention provides a multimer of MHC class I molecule loaded (covalently or not) with the above-mentioned peptide.
  • MHC multimers include MHC dimers, tetramers, pentamers, octamers, etc. (reviewed in Bakker and Schumacher, Current Opinion in Immunology 2005, 17:428-433). MHC multimers are useful, for example, for the detection and purification of antigen-specific T cells.
  • the present invention provides a cell (e.g., a host cell), in an embodiment an isolated cell, comprising the above-mentioned nucleic acid or vector.
  • the present invention provides a cell expressing at its cell surface an MHC molecule (e.g., a HLA-B8 molecule, such as a HLA-B*0801 molecule, and/or an HLA-A3 molecule, such as a HLA-A*0301 molecule and/or an HLA-B44 allele such as HLA-B*4403) loaded with the above-mentioned peptide.
  • the host cell is a primary cell, a cell line or an immortalized cell.
  • the cell is an antigen-presenting cell (APC).
  • APC antigen-presenting cell
  • Nucleic acids and vectors can be introduced into cells via conventional transformation or transfection techniques.
  • transformation and “transfection” refer to techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection and viral-mediated transfection. Suitable methods for transforming or transfecting host cells can for example be found in Sambrook et al. (supra), and other laboratory manuals. Methods for introducing nucleic acids into mammalian cells in vivo are also known, and may be used to deliver the vector DNA of the invention to a subject for gene therapy.
  • the present invention provides T cell receptor (TCR) molecules capable of interacting with the above-mentioned MHC molecule/peptide (MiHA) complex, and nucleic acid molecules encoding such TCR molecules.
  • TCR T cell receptor
  • a TCR according to the present invention will preferably be capable of specifically interacting with an MiHA of the present invention loaded on an MHC molecule, preferably at the surface of a living cell in vitro or in vivo.
  • T cell receptors and in particular nucleic acids encoding TCR's according to the invention may for instance be applied to transfer a TCR from one T cell to another T cell and generate new T cell clones capable of specifically recognizing the MiHA.
  • T cell clones may be provided that essentially are of the genetic make-up of an allogeneic donor, for instance a donor of lymphocytes.
  • the method to provide T cell clones capable of recognizing an MiHA according to the invention may be generated for and can be specifically targeted to tumor cells expressing the MiHA in a graft recipient, preferably an ASCT and/or donor lymphocyte infusion (DLI) recipient subject.
  • a graft recipient preferably an ASCT and/or donor lymphocyte infusion (DLI) recipient subject.
  • the invention provides CD8 T lymphocytes encoding and expressing a T cell receptor capable of interacting with the above-mentioned peptide/MHC molecule complex.
  • Said T lymphocyte may be a recombinant or a naturally selected T lymphocyte.
  • CD8 T lymphocytes of the invention may also be used for or in the methods and pharmaceutical compositions (see below).
  • This specification thus provides at least two methods for producing CD8 T lymphocytes of the invention, comprising the step of bringing undifferentiated lymphocytes into contact with a peptide/MHC molecule complex (typically expressed at the surface of cells, such as APCs) under conditions conducive of triggering an immune response, which may be done in vitro or in vivo for instance in a patient receiving a graft.
  • a peptide/MHC molecule complex typically expressed at the surface of cells, such as APCs
  • a gene encoding the TCR specific for interacting with a peptide/MHC molecule complex which may be obtained from a cell obtained from the previous method or from a subject exhibiting an immune response against peptide/MHC molecule complex, into a host cell and/or a host lymphocyte obtained from a graft recipient or graft donor, and optionally differentiate to cytotoxic T lymphocytes (CTLs).
  • CTLs cytotoxic T lymphocytes
  • MiHA-based cancer immunotherapy is significant.
  • hematologic cancers e.g., leukemia
  • the use of anti-MiHA T cells may replace conventional AHCT because it may provide superior anti-leukemic activity without causing GVHD.
  • GVHD/GVT risk/reward
  • High-avidity T cell responses capable of eradicating tumors can be generated in an allogeneic setting.
  • ASCT allogeneic HLA-matched hematopoietic stem cell transplantation
  • GVT T cell-mediated graft-versus-tumor
  • Immunotherapy in an allogeneic setting enables induction of effective T cell responses due to the fact that T cells of donor origin are not selected for low reactivity against self-antigens of the recipient. Therefore, high-affinity T cells against tumor- or recipient-specific antigens can be found in the T cell inoculum administered to the patient during or after ASCT.
  • the main targets of the tumor-reactive T cell responses are polymorphic proteins for which donor and recipient are disparate, namely MiHAs.
  • the MiHA peptide sequences identified herein may be used for the production of synthetic peptides to be used i) for in vitro priming and expansion of MiHA-specific T cells to be injected into transplant (AHCT) recipients and/or ii) as vaccines to boost the graft-vs.-tumor effect (GVTE) in recipients of MiHA-specific T cells, subsequent to the transplantation.
  • the present invention provides the use of the above-mentioned peptide of sequences (I) to (VII) in the immunotherapy of cancer.
  • the present invention provides a method of treating cancer, said method comprising administering to a subject in need thereof an effective amount of CD8 T lymphocytes recognizing a MHC class I molecule loaded with the above-mentioned peptide.
  • the present invention provides the use of CD8 T lymphocytes recognizing a MHC class I molecule loaded with the above-mentioned peptide for treating cancer in a subject.
  • the present invention provides the use of CD8 T lymphocytes recognizing a MHC class I molecule loaded with the above-mentioned peptide for the preparation/manufacture of a medicament for treating cancer in a subject.
  • the subject is a transplant (e.g., AHCT) recipient.
  • the above-mentioned method or use further comprises determining whether said subject expresses a CENPF nucleic acid comprising a T or a C at a position corresponding to nucleotide 4409 in the nucleic acid sequence of human CENPF ( FIGS. 1A to 1C , NCBI Reference Sequence: NM_016343.3), and/or a CENPF polypeptide comprising a leucine or serine residue at a position corresponding to residue 1412 in the protein sequence of human CENPF ( FIG.
  • the above-mentioned method or use further comprises determining whether said subject expresses a ZWINT nucleic acid comprising an A or a G at a position corresponding to nucleotide 596 in the nucleic acid sequence of human ZWINT ( FIG. 2A , NCBI Reference Sequence: NM_007057.3), and/or a ZWINT polypeptide comprising a arginine or glycine residue at a position corresponding to residue 187 in the protein sequence of human ZWINT ( FIG.
  • the above-mentioned method further comprises determining whether said subject expresses a MTCH2 nucleic acid comprising a C or a G at a position corresponding to nucleotide 1057 in the nucleic acid sequence of human MTCH2 ( FIG. 3A , NCBI Reference Sequence: NM_014342.3), and/or a MTCH2 polypeptide comprising a proline or alanine residue at a position corresponding to residue 290 in the protein sequence of human MTCH2 ( FIG.
  • the above-mentioned method further comprises determining whether said subject expresses an ELF1 nucleic acid comprising an A or T at a position corresponding to nucleotide 1400 in the nucleic acid sequence of human ELF1 ( FIGS. 4A and 4B , NCBI Reference Sequence: NM_172373.3) and/or an ELF1 polypeptide having a threonine or a serine at a position corresponding to residue 343 in the ELF1 protein sequence ( FIG.
  • the above-mentioned method further comprises determining whether said subject expresses an NQO1 nucleic acid comprising an C or T at a position corresponding to nucleotide 615 in the nucleic acid sequence of human NQO1 ( FIG. 5A , NCBI Reference Sequence: NM_000903.2) and/or an NQO1 polypeptide having an arginine or a tryptophan at a position corresponding to residue 139 in the NQO1 protein sequence ( FIG.
  • NCBI Reference Sequence: NP_000894.1 wherein (a) if said subject expresses a NQO1 nucleic acid comprising an C at a position corresponding to nucleotide 615 in the nucleic acid sequence of human NQO1 and/or a NQO1 polypeptide comprising an arginine residue at a position corresponding to residue 139 in the protein sequence of human NQO1, X 17 is R in said peptide; (b) if said subject expresses an NQO1 nucleic acid comprising a T at a position corresponding to nucleotide 615 in the nucleic acid sequence of human NQO1 and/or a NQO1 polypeptide comprising a tryptophan residue at a position corresponding to residue 139 in the protein sequence of human NQO1, X 17 is W in said peptide.
  • the above-mentioned method further comprises determining whether said subject expresses a KIAA0226L nucleic acid comprising a G or A at a position corresponding to nucleotide 1059 in the nucleic acid sequence of human KIAA0226L ( FIGS. 6A and 6B , NCBI Reference Sequence: NM_025113.2) and/or an KIAA0226L polypeptide having a glycine or an arginine at a position corresponding to residue 152 in the KIAA0226L protein sequence ( FIG.
  • the above-mentioned method further comprises determining whether said subject expresses an RMDN1 nucleic acid comprising an A or C at a position corresponding to nucleotide 316 in the nucleic acid sequence of human RMDN1 ( FIG. 7A , NCBI Reference Sequence: NM_016033.2) and/or an RMDN1 polypeptide having a lysine or an asparagine at a position corresponding to residue 52 in the RMDN1 protein sequence ( FIG.
  • nucleic acid and/or protein of interest e.g., CENPF, ZWINT, MTCH2, ELF1, NQO1, KIAA0226L or RMDN1
  • CENPF CENPF
  • ZWINT MTCH2
  • ELF1, NQO1, KIAA0226L or RMDN1 ELF1, NQO1, KIAA0226L or RMDN1
  • suitable methods for detecting alterations at the nucleic acid level include sequencing of the nucleic acid sequence of the nucleic acid of interest (e.g., CENPF, ZWINT, MTCH2, ELF1, NQO1, KIAA0226L or RMDN1); hybridization of a nucleic acid probe capable of specifically hybridizing to a nucleic acid of interest (e.g., CENPF, ZWINT, MTCH2, ELF1, NQO1, KIAA0226L or RMDN1) comprising the polymorphism (the first allele) and not to (or to a lesser extent to) a corresponding nucleic acid that do not comprise the polymorphism (the second allele) (under comparable hybridization conditions, such as stringent hybridization conditions), or vice-versa; restriction fragment length polymorphism analysis (RFLP); Amplified fragment length polymorphism PCR (AFLP-PCR); amplification of a nucleic acid fragment using a primer specific for one of the allele, wherein the
  • nucleic acids of interest e.g., CENPF, ZWINT, MTCH2, ELF1, NQO1, KIAA0226L or RMDN1 nucleic acids
  • PCR polymerase chain reaction
  • the design of various primers for such amplification is known in the ad.
  • the nucleic acid (mRNA) may also be reverse transcribed into cDNA prior to analysis.
  • suitable methods for detecting alterations/polymorphisms at the polypeptide level include sequencing of the polypeptide of interest (e.g., CENPF, ZWINT, MTCH2, ELF1, NQO1, KIAA0226L or RMDN1); digestion of the polypeptide followed by mass spectrometry or HPLC analysis of the peptide fragments, wherein the alteration/polymorphism of the polypeptide of interest (e.g., CENPF, ZWINT, MTCH2, ELF1, NQO1, KIAA0226L or RMDN1) results in an altered mass spectrometry or HPLC spectrum as compared to the native polypeptide of interest (e.g., CENPF, ZWINT, MTCH2, ELF1, NQO1, KIAA0226L or RMDN1); and immunodetection using an immunological reagent (e.g., an antibody, a ligand) which exhibits altered immunoreactivity with a polypeptide comprising the alteration (
  • Immunodetection can measure the amount of binding between a polypeptide molecule and an anti-protein antibody by the use of enzymatic, chromodynamic, radioactive, magnetic, or luminescent labels which are attached to either the anti-protein antibody or a secondary antibody which binds the anti-protein antibody.
  • other high affinity ligands may be used.
  • Immunoassays which can be used include e.g. ELISAs, Western blots, and other techniques known to those of ordinary skill in the art (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999 and Edwards R, Immunodiagnostics: A Practical Approach, Oxford University Press, Oxford; England, 1999).
  • microarrays may also be employed in the format of microarrays, protein-arrays, antibody microarrays, tissue microarrays, electronic biochip or protein-chip based technologies (see Schena M., Microarray Biochip Technology, Eaton Publishing, Natick, Mass., 2000).
  • nucleic acids of interest may be amplified using known methods (e.g., polymerase chain reaction [PCR]) prior to or in conjunction with the detection methods noted herein.
  • PCR polymerase chain reaction
  • the above determining comprises sequencing in a biological sample from a subject a region of a nucleic acid corresponding to the region encompassing (i) nucleotide 4409 of a human CENPF nucleic acid (SEQ ID NO:1), (ii) nucleotide 596 in the nucleic acid of a human ZWINT nucleic acid (SEQ ID NO:3), (iii) nucleotide 1057 in the nucleic acid of a human MTCH2 nucleic acid (SEQ ID NO:5), (iv) nucleotide 1400 in the nucleic acid of a human ELF1 nucleic acid (SEQ ID NO:7), (v) nucleotide 615 in the nucleic acid of a human NQO1 nucleic acid (SEQ ID NO:9), (vi) nucleotide 1059 in the nucleic acid of a human KIAA0226L nucleic acid (SEQ ID NO:11) and/or (vii)
  • the above-mentioned CD8 T lymphocytes are in vitro expanded CD8 T lymphocytes.
  • Expanded CD8 T lymphocytes may be obtained by culturing primary CD8 T lymphocytes (from a donor) under conditions permitting the proliferation and/or differentiation of the CD8 T lymphocytes. Such conditions typically include contacting the CD8 T lymphocytes with cells, such as APCs, expressing at their surface peptide/MHC complexes, in the presence of growth factors and/or cytokines such as IL-2, IL-7 and/or IL-15 (see, e.g., Montes et al., Clin Exp Immunol. 2005 November; 142(2):292-302). Such expanded CD8 T lymphocytes are then administered to the recipient, for example through intravenous infusion.
  • the subject is an allogeneic stem cell transplantation (ASCT) or donor lymphocyte infusion (DLI) recipient.
  • ASCT allogeneic stem cell transplantation
  • DAI donor lymphocyte infusion
  • the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising (a) determining whether a candidate donor expresses a CENPF nucleic acid comprising a T or a C at a position corresponding to nucleotide 4409 in the nucleic acid sequence of human CENPF ( FIGS. 1A to 1D , NCBI Reference Sequence: NM_016343.3), and/or a CENPF polypeptide comprising a leucine or serine residue at a position corresponding to residue 1412 in the protein sequence of human CENPF ( FIG.
  • the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising: (a) determining whether a candidate donor expresses a ZWINT nucleic acid comprising an A or a G at a position corresponding to nucleotide 596 in the nucleic acid sequence of human ZWINT ( FIG. 2A , NCBI Reference Sequence: NM_007057.3), and/or a ZWINT polypeptide comprising a arginine or glycine residue at a position corresponding to residue 187 in the protein sequence of human ZWINT ( FIG.
  • the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising: (a) determining whether a candidate donor expresses a MTCH2 nucleic acid comprising a C or a G at a position corresponding to nucleotide 1057 in the nucleic acid sequence of human MTCH2 ( FIG. 3A , NCBI Reference Sequence: NM_014342.3), and/or a MTCH2 polypeptide comprising a proline or alanine residue at a position corresponding to residue 290 in the protein sequence of human MTCH2 ( FIG.
  • the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising: (a) determining whether a candidate donor expresses an ELF1 nucleic acid comprising an A or T at a position corresponding to nucleotide 1400 in the nucleic acid sequence of human ELF1 ( FIGS. 4A and 4B , NCBI Reference Sequence: NM_172373.3) and/or an ELF1 polypeptide having a threonine or a serine at a position corresponding to residue 343 in the ELF1 protein sequence ( FIG.
  • the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising: (a) determining whether said subject expresses an NQO1 nucleic acid comprising a C or T at a position corresponding to nucleotide 615 in the nucleic acid sequence of human NQO1 ( FIG. 5A , NCBI Reference Sequence: NM_000903.2) and/or an NQO1 polypeptide having an arginine or a tryptophan at a position corresponding to residue 139 in the NQO1 protein sequence ( FIG.
  • the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising: (a) determining whether said subject expresses a KIAA0226L nucleic acid comprising a G or A at a position corresponding to nucleotide 1059 in the nucleic acid sequence of human KIAA0226L ( FIGS. 6A and 6B , NCBI Reference Sequence: NM_025113.2) and/or a KIAA0226L polypeptide having a glycine or an arginine at a position corresponding to residue 152 in the KIAA0226L protein sequence ( FIG.
  • the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising: (a) determining whether said subject expresses an RMDN1 nucleic acid comprising an A or C at a position corresponding to nucleotide 316 in the nucleic acid sequence of human RMDN1 ( FIG. 7A , NCBI Reference Sequence: NM_016033.2) and/or an RMDN1 polypeptide having a lysine or an asparagine at a position corresponding to residue 52 in the RMDN1 protein sequence ( FIG.
  • the above-mentioned cancer comprises tumor cells expressing CENPF, ZWINT, MTCH2, ELF1, NQO1, KIAA0226L and/or RMDN1.
  • the above-mentioned cancer comprises tumor cells expressing CENPF, and is a hematopoietic cancer, such as leukemia, lymphoma, or myeloma, or a solid tumor such as head and neck squamous cell carcinomas, breast cancer, non-Hodgkin's lymphoma or gastrointestinal cancer.
  • a hematopoietic cancer such as leukemia, lymphoma, or myeloma
  • a solid tumor such as head and neck squamous cell carcinomas, breast cancer, non-Hodgkin's lymphoma or gastrointestinal cancer.
  • the above-mentioned cancer comprises tumor cells expressing ZWINT, and is a breast, prostate or bladder cancer.
  • the above-mentioned cancer comprises tumor cells expressing MTCH2, and is a solid tumor/cancer, in a further embodiment lung, thyroid, liver, esophagus, colon or breast cancer, or osteosarcoma.
  • the above-mentioned cancer comprises tumor cells expressing ELF1, and is leukemia, lung cancer (e.g., non-small cell lung cancer), breast cancer or ovarian cancer.
  • lung cancer e.g., non-small cell lung cancer
  • breast cancer e.g., breast cancer or ovarian cancer.
  • the above-mentioned cancer comprises tumor cells expressing NQO1, and is lung cancer (e.g., non-small cell lung cancer), skin cancer, breast cancer, liver cancer (e.g., intrahepatic cholangiocarcinoma) digestive tract cancer such as colorectal cancer, or pancreatic cancer.
  • lung cancer e.g., non-small cell lung cancer
  • skin cancer e.g., skin cancer
  • breast cancer e.g., liver cancer (e.g., intrahepatic cholangiocarcinoma) digestive tract cancer
  • pancreatic cancer e.g., pancreatic cancer.
  • the above-mentioned cancer comprises tumor cells expressing KIAA0226L, and is a lymphoma (e.g., Burkitt's lymphoma), skin cancer, breast cancer, liver cancer (e.g., intrahepatic cholangiocarcinoma) digestive tract cancer such as colorectal cancer, or pancreatic cancer.
  • a lymphoma e.g., Burkitt's lymphoma
  • skin cancer e.g., breast cancer
  • liver cancer e.g., intrahepatic cholangiocarcinoma
  • digestive tract cancer such as colorectal cancer, or pancreatic cancer.
  • PBMCs Peripheral blood mononuclear cells
  • EBV Epstein-Barr virus
  • B-LCLs B lymphoblastoid cell lines
  • Established B-LCLs were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 25 mM of HEPES, 2 mM L-glutamine and penicillin-streptomycin (all from Invitrogen).
  • Transcriptome libraries were generated from 1 ⁇ g of total RNA using the TruSeqTM RNA Sample Prep Kit (v2) (RS-930-1021, Illumina) following the manufacturer's protocol. Briefly, poly-A mRNA was purified using poly-T oligo-attached magnetic beads using two rounds of purification. During the second elution of the poly-A RNA, the RNA was fragmented and primed for cDNA synthesis. Reverse transcription (RT) of the first strand was done using random primers and SuperScriptTM II (InvitroGene).
  • RT Reverse transcription
  • a second round of RT was also done to generate a double-stranded cDNA, which was then purified using Agencourt AMpureTM XP PCR purification system (Beckman Coulter). End repair of fragmented cDNA, adenylation of the 3′ ends and ligation of adaptors were done following the manufacturer's protocol. Enrichment of DNA fragments containing adapter molecules on both ends was done using 15 cycles of PCR amplification and the IlluminaTM PCR mix and primers cocktail.
  • Genomic libraries were constructed from 1 ⁇ g of genomic DNA using the TruSeqTM DNA Sample Preparation Kit (v2) (FC-930-1021, Illumina) following the manufacturer's protocol. This included the following steps: DNA shearing using a CovarisTM S2 instrument, DNA end repair, 3′ ends adenylation, ligation of adaptors, purification of ligation products and PCR amplification to enrich DNA fragments that have adapter molecules.
  • DNA-Seq libraries were subjected to an enrichment step to sequence only the coding portion (exome) of the human genome. 500 ng of DNA-Seq libraries were used for hybrid selection-based exome enrichment with the TruSeqTM exome enrichment kit (FC-930-1012, Illumina) according to the manufacturer's instructions.
  • RNA-Seq Whole Transcriptome Sequencing (RNA-Seq) and Exome Sequencing.
  • RNA-Seq or four exomes libraries were sequenced per lane (8 lanes per slide). Detail of the Illumina sequencing technologies can be found at http://www.illumina.com/applications/detail/sequencinq/dna_sequencing.ilmn. Briefly, DNA or RNA libraries are incorporated into a fluidic flow cell design with 8 individual lanes. The flow cell surface is populated with capture oligonucleotide anchors, which hybridize the appropriately modified DNA segments of a sequencing library.
  • captured DNA templates are amplified in the flow cell by “arching” over and hybridizing to an adjacent anchor oligonucleotide primer.
  • the sequencing reaction is performed by hybridizing a primer complementary to the adapter sequence and then cyclically adding DNA polymerase and a mixture of 4 differently colored fluorescent reversible dye terminators to the captured DNA in the flow cell.
  • unmodified DNA fragments and unincorporated nucleotides are washed away, while captured DNA fragments are extended one nucleotide at a time.
  • the flow cell is scanned, and digital images are acquired to record the locations of fluorescently labeled nucleotide incorporations. Following imaging, the fluorescent dye and the terminal 3 blocker are chemically removed from the DNA before the next nucleotide coupling cycle.
  • Sequence data were mapped to the human reference genome (hg19) using the Ilumina CasavaTM 1.8.1 and the ElandTM v2 mapping softwares.
  • the *.bcl files were converted into compressed FASTQ files, following by demultiplexing of separate multiplexed sequence runs by index.
  • single reads were aligned to the human reference genome using the multiseed and gapped alignment method.
  • Multiseed alignment works by aligning the first seed of 32 bases and consecutive seeds separately. Gapped alignment extends each candidate alignment to the full length of the read and allows for gaps up to 10 bases.
  • a read has to have at least one seed that matches with at most 2 mismatches, and for that seed no gaps are allowed and ii) for the whole read any number of gaps were allowed, as long as they correct at least five mismatches downstream.
  • a probability score was calculated. This score is based on the sequencing base quality values and the positions of the mismatches.
  • the alignment score of a read which is expressed on the Phred scale, was computed from the probability scores of the candidate alignments. The best alignment for a given read corresponded to the candidate alignment with the highest probability score and it was picked if the alignment score exceeded a threshold. Reads that mapped at 2 or more locations (multireads) were not included in further analyses. An additional alignment was done against splice junctions and contaminants (mitochondrial and ribosomal RNA).
  • the first step of the process consists of retrieving the list of all single nucleotide variations (SNV) observed between the reference genome (GRCh37.p2, NCBI) and the sequenced transcriptome of each of our subjects.
  • SNV single nucleotide variations
  • Casava calculates and retrieves statistics about every observed SNV including its position, the reference base, the raw counts for each base, the most probable genotype (max_gt) and the probability of the most probable genotype (Qmax_gt).
  • SNVs called by CasavaTM only those that had a high confidence (Qmax_gt value>20) were considered. SNVs with Qmax_gt value below this threshold were assigned with the reference base instead. This strategy was used to identify SNVs at the transcript level between each of our subjects and the reference genome.
  • MHC class I-associated peptides were released by mild acid treatment, pretreated by desalting with an HLB cartridge, filtered with a 3000 Da cut-off column and separated by cation exchange chromatography (SCX) using an off-line 1100 series binary LC system (Agilent Technologies) as previously described (Fortier et al., 2008).
  • SCX cation exchange chromatography
  • Peptides were loaded at 8 uL/min on a homemade strong cation exchange (SCX) column (0.3 mm internal diameter ⁇ 50 mm length) packed with SCX bulk material (Polysulfoethyl ATM, PolyLC).
  • Peptides were separated into five fractions using a linear gradient of 0-25% B in 25 min (solvent A: 5 mM ammonium formate, 15% acetonitrile, ACN, pH 3.0; solvent B: 2 M ammonium formate, 15% ACN, pH 3.0) and brought to dryness using a Speedvac.
  • MHC class I-associated peptide fractions were resuspended in 2% aqueous ACN (0.2% formic acid) and analyzed by LC-MS/MS using an EksigentTM LC system coupled to a LTQ-OrbitrapTM mass spectrometer (Thermo Electron) (Fortier et al., 2008; de Verteuil et al., 2010; Caron et al., 2011).
  • Peptides were separated in a custom C 18 reversed phase column (150 ⁇ m i.d. ⁇ 100 mm, Jupiter ProteoTM 4 ⁇ m, Phenomenex) at a flow rate of 600 nL/min using a linear gradient of 3-60% aqueous ACN (0.2% formic acid) in 69 mins.
  • Full mass spectra were acquired with the OrbitrapTM analyzer operated at a resolving power of 60 000 (at m/z 400) and collision-activated dissociation tandem mass spectra were acquired in data-dependent mode with the linear ion trap analyzer.
  • Mass calibration used an internal lock mass (protonated (Si(CH 3 ) 2 O)) 6 ; m/z 445.120029) and mass accuracy of peptide measurements was within 5 ppm.
  • Mass spectra were analyzed using XcaliburTM software and peak lists were generated using MascotTM distiller version 2.1.1 (Matrix Science, http://www.matrixscience.com).
  • Database searches were performed against a non-redundant human Uniprot database (containing 110,361 sequences, released on 28 Jul. 2011) (version 101 of the UniProt Gene Ontology Annotation available from the European Bioinformatics Institute (EBI), an academic research institute located on the Wellcome Trust Genome Campus in Hinxton near Cambridge (UK), part of the European Molecular Biology Laboratory; http://www.ebi.ac.uk/Information/. Magrane et al., Database Vol. 2011, Article ID bar009; The UniProt Consortium, Nucleic Acids Res.
  • EBI European Bioinformatics Institute
  • Raw data files were converted to peptide maps comprising m/z values, charge state, retention time and intensity for all detected ions above a threshold of 8000 counts using in-house software (Proteoprofile) (Fortier et al., 2008; de Verteuil et al., 2010; Caron et al., 2011).
  • Peptide maps were aligned together and peptide ions of each map (including non-sequenced ions) were aggregated to their corresponding Mascot identification, creating a peptide abundance profile. When multiple Mascot identifications were associated to the same ions, only the one with the highest score was kept. The intensity counts were then summed for identical peptide sequences resulting in a non-redundant abundance profile of identified peptide sequences.
  • Peptides were filtered by their length and those peptides with the canonical MHC I-associated peptide length (typically 8-11 mers) were kept. Peptides were considered to be undetected/not expressed if they were absent in 3 replicates per subject, and detected/expressed if they were identified in at least 2 replicates per subject.
  • the predicted binding affinity (IC 50 ) of peptides to the allelic products was obtained using NetMHCcons version 1.0 (http://www.cbs.dtu.dk/services/NetMHCcons/) (Karosiene et al., 2011) and was used to classify MHC I peptides. Peptides with an IC 50 below 50 nM were considered as strong binders and peptides with an IC 50 between 50 and 500 nM were considered as weak binders.
  • MiHA peptides were selected according to the following criteria:
  • RNA (cDNA) and DNA sequences encoding MiHAs candidates were manually inspected using the Integrative Genomics Viewer v2.0 (The Broad Institute).
  • the UCSC Repeat Masker track was included to discard candidates that corresponded to repetitive regions.
  • a dbSNP (build 135) track was also used to identify those SNVs in the MiHA-encoding region that corresponded to reported SNPs.
  • MiHA candidates were further inspected for mass accuracy and MS/MS spectra were validated manually using the XcaliburTM software (Thermo Xcalibur 2.2 SP1.48 version).
  • MiHAs are i) peptides ii) whose presence depends on genetic polymorphisms, it was reasoned that high-throughput MiHA discovery would involve a combination of MS and personalized whole transcriptome and/or exome sequencing. Genomic data alone are insufficient for MiHA discovery because even if a genetic polymorphism is identified in a gene, i) only 0.1% of expressed peptides are presented as MAPs at the cell surface and ii) the effect of polymorphisms in trans cannot be predicted (5;6). In MS analyses, peptides are identified through database search with softwares such as Mascot. These databases contain the translated genome of reference and do not include genetic polymorphisms. Since MiHAs are the result of genetic polymorphisms, they cannot be discovered by standard MS alone.
  • a novel method for human MiHA discovery was developed.
  • One of the key elements of the method is the inclusion of personalized translated transcriptome in the database used for peptide identification by MS.
  • PBMCs were isolated from blood samples of 2 HLA-identical siblings who are HLA-A*0301/2902, HLA-B*0801/*4403, HLA-C*0701/*1601.
  • Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (B-LCLs) were derived from PBMCs with Ficoll-PaqueTM Plus. Three biological replicates of 4 ⁇ 10 8 exponentially growing B-LCLs were prepared from each subject.
  • MAPs were released by mild acid treatment and separated by cation exchange chromatography MAP fractions were analyzed by LC-MS/MS.
  • RNA-seq provided a most useful complementary dataset since i) it can highlight differences in transcript levels between two subjects (due for instance to polymorphisms in promoter regions), and ii) it covers unannotated genes or pseudogenes not covered by the exome capture protocol.
  • the latter genes can be a source of MAPs and may have a significant contribution to the MiHA landscape.
  • the translated exome-transcriptome were then used as a personalized Mascot protein sequence database, helping to retrieve MAPs overlapping variant locations and most importantly documenting differences in the MAP repertoire of HLA-identical siblings: a MAP present in only one of two HLA-identical siblings is an MiHA.
  • Allelic MiHAs comprising the amino acid sequence ELQEKF L SL vs. ELQEKF S SL have been identified. These MiHAs are presented at the cell surface by the MHC class I allele HLA-B*0801. These peptides are not listed in the Immune Epitope Database (Vita et al., 2010) which includes a repertoire of all well characterized peptides presented by MHC molecules (HLA in humans). These MiHAs derive from a single nucleotide polymorphism in the centromer protein F, 350/400 kDa (mitosin) (CENPF) gene.
  • This single nucleotide polymorphism is listed as rs3795517 in the dbSNP database (Sherry et al., 2001) (http://www.ncbi.nlm.nih.gov/projects/SNP/). Two alleles are found at this locus; one codes for a CENPF protein containing the ELQEKF L SL sequence, and the other codes for a CENPF protein with the ELQEKF S SL sequence.
  • the single nucleotide polymorphism corresponds to a T to C substitution at a position corresponding to nucleotide 4409 in the nucleic acid sequence of human CENPF ( FIGS.
  • CENPF is a transient kinetochore protein that plays multiple roles in cell division. CENPF expression increases in the G2 phase but it is rapidly proteolyzed at the end of mitosis.
  • the peptide of the sequence (I) described herein may be used in the immunotherapy of one or more of these cancers.
  • the amino acid sequence of another pair of MiHAs identified herein is QELD G VFQKL vs. QELD R VFQKL. These MiHAs are presented at the cell surface by HLA-B*4403 molecules. These peptides are not listed in the Immune Epitope Database (Vita et al., 2010). These MiHAs derive from a single nucleotide polymorphism in the ZWINT (ZW10 interactor) gene. This single nucleotide polymorphism is listed as rs2241666 in the dbSNP database (Sherry et al., 2001).
  • the single nucleotide polymorphism corresponds to an A to G substitution at a position corresponding to nucleotide 596 in the nucleic acid sequence of human ZWINT ( FIG. 2A , NCBI Reference Sequence: NM_007057.3), leading to an arginine to glycine substitution at a position corresponding to residue 187 in the ZWINT protein sequence ( FIG. 2B , NCBI Reference Sequence: NP_008988.2).
  • ZWINT is a known component of the kinetochore complex that is required for the mitotic spindle checkpoint. Overexpression of ZWINT has been observed in several types of cancer (notably breast, prostate and bladder) and correlates with an increased proliferation of cancer cells (Endo et al., 2012; Ho et al., 2012; Urbanucci et al., 2012). Also, according to the EMBL-EBI Gene Expression Atlas (Kapushesky et al., Nucl. Acids Res .
  • the peptide of the sequence (II) described herein may be used in the immunotherapy of one or more of these cancers.
  • the amino acid sequence of a third pair of MiHAs identified herein is SLFFRKV P F vs. SLFFRKV A F. These MiHAs are presented at the cell surface by HLA-B*0801 molecules. These peptides are not listed in the Immune Epitope Database (Vita et al., 2010). These MiHAs derive from a single nucleotide polymorphism in the MTCH2 (mitochondrial carrier homologue 2) gene. This single nucleotide polymorphism is listed as rs1064608 in the dbSNP database (Sherry et al., 2001).
  • the single nucleotide polymorphism corresponds to a C to G substitution at a position corresponding to nucleotide 1057 in the nucleic acid sequence of human MTCH2 ( FIG. 3A , NCBI Reference Sequence: NM_014342.3), leading to a proline to alanine substitution at a position corresponding to residue 290 in the MTCH2 protein sequence ( FIG. 3B , NCBI Reference Sequence: NP_055157.1).
  • MTCH2 interact with proapoptotic truncated BID and thereby regulate apoptosis, and is upregulated/involved in several types of cancer, notably solid tumors (lung, thyroid, liver, esophagus, colon, breast) and osteosarcoma (Yu et al., 2008, Grinberg et al., 2005; Katz et al., 2012). Also, according to the EMBL-EBI Gene Expression Atlas (Kapushesky et al., Nucl. Acids Res . (2012) 40 (D1): D1077-D1081), overexpression of MTCH2 was detected in lymphomas, leukemias and myelomas. Accordingly, in an embodiment, the peptide of the sequence (III) described herein may be used in the immunotherapy of one or more of these cancers.
  • the amino acid sequence of a fourth pair of MiHAs identified herein is S VLKPGNSK vs. T VLKPGNSK. These MiHAs are presented at the cell surface by HLA-A*0301 molecules. These peptides are not listed in the Immune Epitope Database (Vita et al., 2010). These MiHAs derive from a single nucleotide polymorphism in the ELF1 [E74-like factor 1 (ets domain transcription factor)] gene. This single nucleotide polymorphism is listed as rs1056820 in the dbSNP database (Sherry et al., 2001).
  • the single nucleotide polymorphism corresponds to an A to T substitution at a position corresponding to nucleotide 1400 in the nucleic acid sequence of human ELF1 ( FIGS. 4A and 4B , NCBI Reference Sequence: NM_172373.3), leading to a threonine to serine substitution at a position corresponding to residue 343 in the ELF1 protein sequence ( FIG. 4C , NCBI Reference Sequence: NP_758961.1).
  • ELF1 is a transcriptional factor that is involved in the transcriptional activation of oncogenic pathways in cancer cells, such as leukemias, non-small cell lung cancer, breast and ovarian cancer (Andrews et al., 2008; Xiang et al., 2010; Yang et al., 2010). Also, according to the EMBL-EBI Gene Expression Atlas (Kapushesky et al., Nucl. Acids Res .
  • the peptide of the sequence (IV) described herein may be used in the immunotherapy of one or more of these cancers.
  • the amino acid sequence of another pair of MiHAs is AMYDKGPF R SK vs. AMYDKGPF W SK. These MiHAs are presented at the cell surface by HLA-A*0301. These peptides are not listed in the Immune Epitope Database (Vita et al., 2010). These MiHAs derive from a single nucleotide polymorphism in the NQO1 [NAD(P)H dehydrogenase, quinone 1] gene. This single nucleotide polymorphism is listed as r51131341 in the dbSNP database (Sherry et al., 2001).
  • the single nucleotide polymorphism corresponds to a C to T substitution at a position corresponding to nucleotide 615 in the nucleic acid sequence of human NQO1 ( FIG. 5A , NCBI Reference Sequence: NM_000903.2), leading to an arginine to tryptophan substitution at a position corresponding to residue 139 in the NQO1 protein sequence ( FIG. 5B , NCBI Reference Sequence: NP_000894.1).
  • NQO1 is a cytosolic enzyme that catalyzes the reduction of various quinones using flavin adenine dinucleotide (FAD) as a cofactor. This protein's enzymatic activity prevents the one electron reduction of quinones that results in the production of radical species.
  • FAD flavin adenine dinucleotide
  • NQO1 Mutations in this NQO1 have been associated with susceptibility to various forms of cancer and altered expression of this NQO1 protein has been seen in many tumors, including non-small cell lung cancer, skin cancer (e.g., melanomas), breast cancer, liver cancer (e.g., intrahepatic cholangiocarcinoma) and digestive tract cancer such as colorectal cancer (Kolesar et al., 2011; Wakai et al., 2011; Jamieson et al., 2011; Ding et al., 2012; Yang et al., 2012; Patrick and Jaiswal, 2012).
  • skin cancer e.g., melanomas
  • breast cancer e.g., liver cancer (e.g., intrahepatic cholangiocarcinoma)
  • digestive tract cancer such as colorectal cancer (Kolesar et al., 2011; Wakai et al., 2011; Jamieson et al., 2011; Ding e
  • NQO1 Substrate was recently shown to possess potent antitumor activity against a wide spectrum of cancer cells, such as pancreatic and lung cancer cells (Huang et al., 2012). Also, according to the EMBL-EBI Gene Expression Atlas (Kapushesky et al., Nucl. Acids Res . (2012) 40 (D1): D1077-D1081), overexpression of NQO1 was detected in lymphomas (Hodgkin's lymphomas, anaplastic large cell lymphoma) and brain cancers (glioblastomas, subependymal giant cell astrocytomas). Accordingly, in an embodiment, the peptide of the sequence (V) described herein may be used in the immunotherapy of one or more of these cancers.
  • the amino acid sequence of another pair of MiHAs is RVSLPTSPG vs. RVSLPTSPR. These MiHAs are presented at the cell surface by HLA-A*0301. These peptides are not listed in the Immune Epitope Database (Vita et al., 2010) which includes a repertoire of all well characterized peptides presented by MHC molecules (HLA in humans).
  • MiHAs derive from a single nucleotide polymorphism in the KIAA0226-like gene (also known as C13orf18).
  • This single nucleotide polymorphism is listed as rs1408184 in the dbSNP database (Sherry et al., 2001). Two alleles are found at this locus; one codes for an KIAA0226L protein containing the RVSLPTSP G sequence, and the other codes for an KIAA0226L protein with the RVSLPTSP R sequence.
  • the single nucleotide polymorphism corresponds to a G to A substitution at a position corresponding to nucleotide 1059 in the nucleic acid sequence of human KIAA0226L ( FIG.
  • FIG. 6A NCBI Reference Sequence: NM_025113.2
  • FIG. 6B NCBI Reference Sequence: NP_079389.2
  • the function of KIAA0226L is largely unknown, and according to the BioGPS database, KIAA0226L is upregulated in B lymphocytes and Burkitt's lymphoma cells. Also, according to the EMBL-EBI Gene Expression Atlas (Kapushesky et al., Nucl. Acids Res .
  • the peptide of the sequence (VI) described herein may be used in the immunotherapy of one or more of these cancers.
  • MiHAs The amino acid sequence of another pair of MiHAs is VMGNPGTF K vs. VMGNPGTF N . These MiHAs are presented at the cell surface by HLA-A*0301. These peptides are not listed in the Immune Epitope Database (Vita et al., 2010) which includes a repertoire of all well characterized peptides presented by MHC molecules (HLA in humans).
  • MiHAs derive from a single nucleotide polymorphism in the RMDN1 (regulator of microtubule dynamics 1) gene (also known as FAM82B).
  • This single nucleotide polymorphism is listed as rs6980476 in the dbSNP database (Sherry et al., 2001). Two alleles are found at this locus; one codes for an RMDN1 protein containing the VMGNPGTF K sequence, and the other codes for an RMDN1 protein with the VMGNPGTF N sequence.
  • the single nucleotide polymorphism corresponds to an A to C substitution at a position corresponding to nucleotide 316 in the nucleic acid sequence of human RMDN1 ( FIG.
  • RMDN1 is a microtubule-associated protein that plays a role in chromosome segregation in Caenorhabditis elegans (Oishi et al., J Cell Biol. 179, 1149-1162, 2007). According to the BioGPS database, RMDN1 is upregulated in B lymphoblasts and Burkitt's lymphoma cells.
  • the peptide of the sequence (VII) described herein may be used in the immunotherapy of one or more of these cancers.

Abstract

A novel method for human minor histocompatibility antigen (MiHA) discovery, novel MiHAs identified using this method, as well as uses of the novel MiHAs, are described. One of the features of the novel method is the inclusion of personalized translated transcriptome and/or exome in the database used for peptide identification by mass spectroscopy (MS). Candidate MiHAs are identified by comparing the personalized transcriptome and/or exome to a reference genome and/or to the transcriptome and/or exome of an HLA-matched subject.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 14/421,310, filed Feb. 12, 2015, which is a 35 U.S.C. § 371 national phase application of PCT/CA2013/050580, filed Jul. 25, 2013, which claims the benefit of U.S. provisional application Ser. No. 61/683,361, filed on Aug. 15, 2012, and of U.S. provisional application Ser. No. 61/818,040, filed on May 1, 2013, which are incorporated herein by reference in their entirety.
TECHNICAL FIELD
The present invention generally relates to histocompatibility antigens, and more specifically to minor histocompatibility antigens (MiHAs), identification and use thereof.
SEQUENCE LISTING
Pursuant to 37 C.F.R. 1.821(c), a sequence listing is submitted herewith as an ASCII complaint text file named “585721 GGT-009USCON Sequence Listing-TXT.txt”, created on Feb. 12, 2015 and having a size of ˜147 kilobytes. The content of the aforementioned file is hereby incorporated by reference in its entirety.
BACKGROUND ART
Histocompatibility antigens are a group of cell membrane alloantigens that are recognized by T lymphocytes and thereby initiate graft rejection or graft-versus-host disease (GVHD) following transplantation (1). In the early days of immunogenetics, the identification of major histocompatibility complex (MHC) antigens was based on their strong immunogenicity in skin transplant experiments between congenic-resistant strains of mice. Other less potent antigens were called minor histocompatibility antigens (MiHA). It soon became obvious that a distinction between major and minor antigens based solely on their immunogenicity was imprecise, as some MHC antigens are weak immunogens while some MiHA appear “neither weak nor minor” (2;3) It is now known that MHC antigens (also referred to as HLA antigens) are transmembrane glycoproteins encoded by closely linked polymorphic loci located on chromosome 6 in humans. Their primary role is to bind endogenous and exogenous peptides that are scrutinized by T cells. MHC (or HLA) molecules present thousands of peptides at the surface of human cells (4;5). These MHC-associated peptides (MAPs) are referred to as the immunopeptidome and they originate from proteasomal processing and further processing of endogenous proteins (6-8). The immunopeptidome of identical twins (also referred to as syngeneic subjects) is identical. By contrast, MAPs present on cells from HLA-identical non-syngeneic subjects are classified into two categories: i) invariant MAPs which are present in all subjects with a given HLA type, and ii) MiHAs which are MAPs present in some but absent in other subjects (9). When T cells are transplanted into an MHC-identical host, they react promptly and specifically to what they see as non-self: host-specific MiHAs. MiHAs are essentially genetic polymorphisms that are immunogenic for T cells. MiHAs are a consequence of any form of accumulated genetic variation that translates to differential MAP display (3;9-13).
Two main strategies can be used for cancer immunotherapy: vaccination and adoptive T-cell immunotherapy (ATCI). The term “ATCI” refers to transfusion of T lymphocytes that may come from different types of donors: the patient (autologous), a genetically-identical twin (syngeneic), or a non-identical donor (allogeneic). To date, ATCI has yielded much higher cancer remission and cure rates than vaccines, and the most widely used form of cancer ATCI is allogeneic hematopoietic cell transplantation (AHCT) (17-22). The graft-versus-tumor (GVT) effect induced by allogeneic AHCT is due mainly to T-cell responses against host MiHAs: GVT is abrogated if the donor is an identical twin (no MiHA differences with the recipient) or if the graft is T-cell depleted (20;23). More than 200,000 individuals treated for hematological malignancies owe their life to the MiHA-dependent GVT effect which represents the most striking evidence of the ability of the human immune system to eradicate neoplasias (18;24-28). Though the allogeneic GVT effect is being used essentially to treat patients with hematologic malignancies, preliminary evidence suggests that it may be also effective for the treatment of solid tumors (29-33). Nonetheless, the considerable potential of MiHA-targeted cancer immunotherapy has not been properly exploited in medicine. In current medical practice, MiHA-based ATCI is limited to “conventional” AHCT, that is, injection of hematopoietic cells from an allogeneic HLA-matched donor. Such unselective injection of allogeneic lymphocytes is a very rudimentary form of MiHA-targeted therapy. First, it lacks specificity and is therefore highly toxic: unselected allogeneic T cells react against a multitude of host MiHAs and thereby induce GVHD in 60% of recipients. GVHD is always incapacitating and frequently lethal (34-38). Second, conventional AHCT induces only an attenuated form of GVT reaction because donor T cells are not being primed (pre-activated) against specific MiHAs expressed on cancer cells prior to injection into the patient. While primed T cells are resistant to tolerance induction, nave T cells can be tolerized by tumor cells (39-42).
It has been demonstrated in mice models of AHCT that, by replacing unselected donor lymphocytes with CD8 T cells primed against a single MiHA, it was possible to cure leukemia and solid tumors without causing GVHD or any untoward effect (33;43;44). Success depends on two main points: selection of an immunodominant (highly immunogenic) MiHA expressed on neoplastic cells, and priming of donor CD8 T cells against the target MiHA prior to AHCT. A recent article (20) describes why MiHA-targeted ATCI is so effective and how translation of this approach in the clinic could have a significant impact on cancer immunotherapy. Implementation of MiHA-targeted ATCI in humans has been limited mainly by the paucity of molecularly defined human MiHAs. Thus, only 33% of patients with leukemia would be eligible for MiHA-based ATCI (15).
Human MiHAs have been discovered using reductionist T-cell based methods. Starting with cytotoxic T lymphocytes (CTLs) from an individual reactive against cells of another HLA-identical subject, investigators have tried and identified MiHAs recognized by these T cells. Different methods have used to do so. First, CTLs were tested on MiHA-negative cells coated with MAPs eluted from MiHA-positive cells. The MAP eluates were fractionated and ultimately the MiHA recognized by CTLs was sequenced by mass spectrometry (MS) (48-53). Second, CTLs were used to screen MiHA-negative cells transfected with cDNA libraries to identify MiHA-coding transcripts (16;54-59). Finally, CTLs have been tested on lymphoblastoid cell lines from many subjects and linkage analyses were performed (based for instance on whole genome association scans or HapMap resources) on lines recognized or not by CTLs (60-67).
The various methods used to discover MiHAs present significant caveats. Firstly, they are not really suitable for high-throughput MiHA discovery: MiHA discovery is made one by one and depends on the availability of a CTL line. Secondly, only MiHAs that have been eluted from living cells and identified by MS can be considered to be validated (direct identification). In the other cases (indirect identification), uncertainty remains as to the exact structure of MiHAs naturally presented at the cell surface (an important criterion for MiHA-targeted immunotherapy). The ambiguity stems mainly from two factors: i) T cells are eminently cross-reactive and can recognize more than one peptide (68); ii) bioinformatic tools used for identification of MAPs in general and MiHAs in particular do not have sufficient reliability to replace direct proteomic identification (69-71).
Thus, there is a need for novel approaches for the identification of MiHAs.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
SUMMARY OF THE INVENTION
In a first aspect, the present invention provides a method of identifying a minor histocompatibility antigen (MiHA) candidate, the method comprising: (a) isolating and sequencing MHC-associated peptides (MAPs) in a first cell sample from a first subject; (b) performing a whole transcriptome and/or exome sequencing on a second cell sample obtained from said first subject; (c) comparing the sequenced whole transcriptome and/or exome to a reference genome to identify single nucleotide variations (SNVs) between the transcriptome and/or exome of said first subject and the reference genome; (d) in silico translating the sequences containing the identified SNVs to identify peptide sequences comprising at least one non-synonymous mutation caused by said SNVs; (e) comparing the sequences of the MAPs isolated in (a) with the peptide sequences identified in (d); and (f) identifying a MiHA candidate based on said comparison.
In another aspect, the present invention provides a method of identifying a minor histocompatibility antigen (MiHA) candidate, the method comprising: (a) isolating and sequencing MHC-associated peptides (MAPs) in a first cell sample from a first and second subjects, wherein said first and second subjects are human leukocyte antigen (HLA)-matched; (b) performing a whole transcriptome and/or exome sequencing on a second cell sample obtained from said first and second subjects; (c) comparing the sequenced whole transcriptomes and/or exomes to identify single nucleotide variations (SNVs) between the transcriptomes and/or exomes of said first and second subjects; (d) in silico translating the sequences containing the identified SNVs to identify peptide sequences comprising at least one non-synonymous mutation caused by said SNVs; (e) comparing the sequences of the MAPs isolated in (a) with the peptide sequences identified in (d); and (f) identifying a MiHA candidate based on said comparison.
In an embodiment, the above-mentioned MiHA candidate is a MAP whose sequence comprises at least one mutation relative to the corresponding sequence translated from the reference genome.
In an embodiment, the above-mentioned MiHA candidate is a MAP present in the first cell sample from said first subject but absent from the first cell sample from said second subject.
In an embodiment, the above-mentioned reference genome is the Genome Reference Consortium Human Build 37 (GRCh37).
In an embodiment, the above-mentioned first and/or second cell sample is a peripheral blood cell sample. In a further embodiment, the above-mentioned peripheral blood cell sample an immortalized peripheral blood cell sample. In a further embodiment, the above-mentioned immortalized peripheral blood cell sample is an Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell line.
In an embodiment, the above-mentioned isolating MAPs comprises (i) releasing said MAPs from said cell sample by mild acid treatment; and (ii) subjecting the released MAPs to chromatography.
In an embodiment, the above-mentioned method further comprises filtering the released peptides with a size exclusion column prior to said chromatography. In a further embodiment, the above-mentioned size exclusion column has a cut-off of about 3000 Da. In an embodiment, the above-mentioned chromatography is cation exchange chromatography.
In an embodiment, the above-mentioned peptide sequences of (d) have a length of 12 amino acids or less. In a further embodiment, the above-mentioned peptide sequences of (d) have a length of 8 to 11 amino acids.
In an embodiment, the above-mentioned comparing comprises subjecting the MAPs isolated in (a) to mass spectrometry and comparing the MS spectra obtained with the peptide sequences identified in (d).
In an embodiment, the above-mentioned method further comprises determining the binding of the MiHA candidate identified in (f) to a major histocompatibility complex (MHC) class I molecule.
In another aspect, the present invention provides a peptide of 50 amino acids or less comprising the sequence (I)
Z1-X1LQEKFX2SX3-Z2 (I)

wherein Z1 is an amino terminal modifying group or is absent; X1 is a sequence of 1 to 43 amino acids or is absent; X2 is L or S; X3 is a sequence of 1 to 43 amino acids or is absent; and Z2 is a carboxy terminal modifying group or is absent. In an embodiment, X1 is an acidic amino acid, in a further embodiment glutamic acid (E). In an embodiment, X3 is an amino acid, in a further embodiment a hydrophobic amino acid, more particularly leucine (L). In an embodiment, X2 is L. In another embodiment, X2 is S. In an embodiment, the peptide comprises the sequence ELQEKFLSL (SEQ ID NO: 15), in a further embodiment the peptide is ELQEKFLSL (SEQ ID NO: 15). In another embodiment, the peptide comprises the sequence ELQEKFSSL (SEQ ID NO: 16), in a further embodiment the peptide is ELQEKFSSL (SEQ ID NO: 16).
In another aspect, the present invention provides a method of treating cancer, said method comprising administering to a subject in need thereof an effective amount of CD8 T lymphocytes recognizing a MHC class I molecule of the HLA-B*0801 allele loaded with the above-mentioned peptide (I). In an embodiment, the above-mentioned method further comprises determining whether said subject expresses a CENPF nucleic acid comprising a T or a C at a position corresponding to nucleotide 4409 in the nucleic acid sequence of human CENPF (FIGS. 1A to 1D, NCBI Reference Sequence: NM_016343.3), and/or a CENPF polypeptide comprising a leucine or serine residue at a position corresponding to residue 1412 in the protein sequence of human CENPF (FIG. 1E, NCBI Reference Sequence: NP_057427.3), wherein (a) if said subject expresses a CENPF nucleic acid comprising a T at a position corresponding to nucleotide 4409 in the nucleic acid sequence of human CENPF and/or a CENPF polypeptide comprising a leucine residue at a position corresponding to residue 1412 in the protein sequence of human CENPF, X2 is L in said peptide; (b) if said subject expresses a CENPF nucleic acid comprising a C at a position corresponding to nucleotide 4409 in the nucleic acid sequence of human CENPF and/or a CENPF polypeptide comprising a serine residue at a position corresponding to residue 1412 in the protein sequence of human CENPF, X2 is S in said peptide.
In an embodiment, the above-mentioned determining comprises sequencing a CENPF nucleic acid. In an embodiment, the above-mentioned CD8 T lymphocytes are in vitro expanded CD8 T lymphocytes.
In an embodiment, the above-mentioned method further comprises: (i) if said subject is the subject of (a), culturing CD8 T lymphocytes from a second subject comprising a C at a position corresponding to nucleotide 4409 in the nucleic acid sequence of human CENPF and/or a CENPF polypeptide comprising a serine residue at a position corresponding to residue 1412 in the protein sequence of human CENPF in the presence of cells expressing a MHC class I molecule of the HLA-B*0801 allele loaded with the above-mentioned peptide (I), wherein X2 is L in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (ii) if said subject is the subject of (b), culturing CD8 T lymphocytes from a second subject comprising a T at a position corresponding to nucleotide 4409 in the nucleic acid sequence of human CENPF and/or a CENPF polypeptide comprising a leucine residue at a position corresponding to residue 1412 in the protein sequence of human CENPF in the presence of cells expressing a MHC class I molecule of the HLA-B*0801 allele loaded with the above-mentioned peptide (I), wherein X2 is S in said peptide, under conditions suitable for CD8 T lymphocyte expansion.
In an embodiment, the above-mentioned subject is an allogeneic stem cell transplantation (ASCT) recipient.
In another aspect, the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising: (a) determining whether a candidate donor expresses a CENPF nucleic acid comprising a T or a C at a position corresponding to nucleotide 4409 in the nucleic acid sequence of human CENPF (FIGS. 1A to 1D, NCBI Reference Sequence: NM_016343.3), and/or a CENPF polypeptide comprising a leucine or serine residue at a position corresponding to residue 1412 in the protein sequence of human CENPF (FIG. 1E, NCBI Reference Sequence: NP_057427.3) and (b)(i) if said candidate donor expresses a CENPF nucleic acid comprising a T at a position corresponding to nucleotide 4409 in the nucleic acid sequence of human CENPF and/or a CENPF polypeptide comprising a leucine residue at a position corresponding to residue 1412 in the protein sequence of human CENPF, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-B*0801 allele loaded with the above-mentioned peptide (I), wherein X2 is S in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (b)(ii) if said candidate donor expresses a CENPF nucleic acid comprising a C at a position corresponding to nucleotide 4409 in the nucleic acid sequence of human CENPF and/or a CENPF polypeptide comprising a serine residue at a position corresponding to residue 1412 in the protein sequence of human CENPF, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-B*0801 allele loaded with the above-mentioned peptide (I), wherein X2 is L in said peptide, under conditions suitable for CD8 T lymphocyte expansion.
In another aspect, the present invention provides a peptide of 50 amino acids or less comprising the sequence (II)
Z1-X4ELDX5VFQX6X7-Z2 (II)

wherein Z1 is an amino terminal modifying group or is absent; X4 is a sequence of 1 to 43 amino acids or is absent; X5 is G or R; X6 is an amino acid or is absent; X7 is a sequence of 1 to 43 amino acids or is absent; and Z2 is a carboxy terminal modifying group or is absent.
In an embodiment, X4 is glutamine (Q). In an embodiment, X6 is an amino acid, in a further embodiment a basic amino acid, more particularly lysine (K). In an embodiment, X7 is an amino acid, in a further embodiment leucine (L). In an embodiment, X5 is G. In another embodiment, X5 is R. In an embodiment, the peptide comprises the sequence QELDGVFQKL (SEQ ID NO:17). In a further embodiment, the peptide is QELDGVFQKL (SEQ ID NO:17). In another embodiment, the peptide comprises the sequence QELDRVFQKL (SEQ ID NO:18). In a further embodiment, the peptide is QELDRVFQKL (SEQ ID NO:18).
In another aspect, the present invention provides a method of treating cancer, said method comprising administering to a subject in need thereof an effective amount of CD8 T lymphocytes recognizing a MHC class I molecule of the HLA-B*4403 allele loaded with the above-mentioned peptide (II).
In an embodiment, the above-mentioned method further comprises determining whether said subject expresses a ZWINT nucleic acid comprising an A or a G at a position corresponding to nucleotide 596 in the nucleic acid sequence of human ZWINT (FIG. 2A, NCBI Reference Sequence: NM_007057.3), and/or a ZWINT polypeptide comprising a arginine or glycine residue at a position corresponding to residue 187 in the protein sequence of human ZWINT (FIG. 2B, NCBI Reference Sequence: NP_008988.2), wherein (a) if said subject expresses a ZWINT nucleic acid comprising an A at a position corresponding to nucleotide 596 in the nucleic acid sequence of human ZWINT and/or a ZWINT polypeptide comprising an arginine residue at a position corresponding to residue 187 in the protein sequence of human ZWINT, X5 is R in said peptide; (b) if said subject expresses a ZWINT nucleic acid comprising a G at a position corresponding to nucleotide 596 in the nucleic acid sequence of human ZWINT and/or a ZWINT polypeptide comprising a glycine residue at a position corresponding to residue 187 in the protein sequence of human ZWINT, X5 is G in said peptide.
In an embodiment, the above-mentioned determining comprises sequencing a human ZWINT nucleic acid. In an embodiment, the above-mentioned CD8 T lymphocytes are in vitro expanded CD8 T lymphocytes.
In an embodiment, the above-mentioned method further comprises: (i) if said subject is the subject of (a), culturing CD8 T lymphocytes from a second subject comprising a G at a position corresponding to nucleotide 596 in the nucleic acid sequence of human ZWINT and/or a ZWINT polypeptide comprising a glycine residue at a position corresponding to residue 187 in the protein sequence of human ZWINT in the presence of cells expressing a MHC class I molecule of the HLA-B*4403 allele loaded with the above-mentioned peptide (II), wherein X5 is R in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (ii) if said subject is the subject of (b), culturing CD8 T lymphocytes from a second subject comprising an A at a position corresponding to nucleotide 596 in the nucleic acid sequence of human ZWINT and/or a ZWINT polypeptide comprising an arginine residue at a position corresponding to residue 187 in the protein sequence of human ZWINT in the presence of cells expressing a MHC class I molecule of the HLA-B*4403 allele loaded with the above-mentioned peptide (II), wherein X5 is G in said peptide, under conditions suitable for CD8 T lymphocyte expansion. In an embodiment, the above-mentioned subject is an allogeneic stem cell transplantation (ASCT) recipient.
In another aspect, the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising: (a) determining whether a candidate donor expresses a ZWINT nucleic acid comprising an A or a G at a position corresponding to nucleotide 596 in the nucleic acid sequence of human ZWINT (FIG. 2A, NCBI Reference Sequence: NM_007057.3), and/or a ZWINT polypeptide comprising a arginine or glycine residue at a position corresponding to residue 187 in the protein sequence of human ZWINT (FIG. 2B, NCBI Reference Sequence: NP_008988.2) and (b)(i) if said candidate donor expresses a ZWINT nucleic acid comprising an A at a position corresponding to nucleotide 596 in the nucleic acid sequence of human ZWINT and/or a ZWINT polypeptide comprising an arginine residue at a position corresponding to residue 187 in the protein sequence of human ZWINT, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-B*4403 allele loaded with the above-mentioned peptide (II), wherein X5 is G in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (b)(ii) if said candidate donor expresses a ZWINT nucleic acid comprising a G at a position corresponding to nucleotide 596 in the nucleic acid sequence of human ZWINT and/or a ZWINT polypeptide comprising a glycine residue at a position corresponding to residue 187 in the protein sequence of human ZWINT, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-B*4403 allele loaded with the above-mentioned peptide (II), wherein X5 is R in said peptide, under conditions suitable for CD8 T lymphocyte expansion.
In another aspect, the present invention provides a peptide of 50 amino acids or less comprising the sequence (III)
Z1-X8LFFRKVX9X10-Z2 (III)

wherein Z1 is an amino terminal modifying group or is absent; X8 is a sequence of 1 to 43 amino acids or is absent; X9 is P or A; X10 is a sequence of 1 to 43 amino acids or is absent; and Z2 is a carboxy terminal modifying group or is absent. In an embodiment, X8 is serine (S). In an embodiment, X10 is an amino acid, in a further embodiment an aromatic amino acid, more particularly phenylalanine (F). In an embodiment, X9 is P. In another embodiment, X9 is A. In an embodiment, the peptide comprises the sequence SLFFRKVPF (SEQ ID NO:19). In a further embodiment, the peptide is SLFFRKVPF (SEQ ID NO:19). In another embodiment, the peptide comprises the sequence SLFFRKVAF (SEQ ID NO:20). In a further embodiment, the peptide is SLFFRKVAF (SEQ ID NO:20).
In another aspect, the present invention provides a method of treating cancer, said method comprising administering to a subject in need thereof an effective amount of CD8 T lymphocytes recognizing a MHC class I molecule of the HLA-B*0801 allele loaded with the above-mentioned peptide.
In an embodiment, the above-mentioned method further comprises determining whether said subject expresses a MTCH2 nucleic acid comprising a C or a G at a position corresponding to nucleotide 1057 in the nucleic acid sequence of human MTCH2 (FIG. 3A, NCBI Reference Sequence: NM_014342.3), and/or a MTCH2 polypeptide comprising a proline or alanine residue at a position corresponding to residue 290 in the protein sequence of human MTCH2 (FIG. 3B, NCBI Reference Sequence: NP_055157.1), wherein (a) if said subject expresses a MTCH2 nucleic acid comprising a C at a position corresponding to nucleotide 1057 in the nucleic acid sequence of human MTCH2 and/or a MTCH2 polypeptide comprising a proline residue at a position corresponding to residue 290 in the protein sequence of human MTCH2, X9 is P in said peptide; (b) if said subject expresses a MTCH2 nucleic acid comprising a G at a position corresponding to nucleotide 1057 in the nucleic acid sequence of human MTCH2 and/or a MTCH2 polypeptide comprising an alanine residue at a position corresponding to residue 290 in the protein sequence of human MTCH2, X5 is A in said peptide.
In an embodiment, the above-mentioned determining comprises sequencing a human MTCH2 nucleic acid. In an embodiment, the above-mentioned CD8 T lymphocytes are in vitro expanded CD8 T lymphocytes.
In an embodiment, the above-mentioned method further comprises: (i) if said subject is the subject of (a), culturing CD8 T lymphocytes from a second subject comprising a G at a position corresponding to nucleotide 1057 in the nucleic acid sequence of human MTCH2 and/or a MTCH2 polypeptide comprising an alanine residue at a position corresponding to residue 290 in the protein sequence of human MTCH2 in the presence of cells expressing a MHC class I molecule of the HLA-B*0801 allele loaded with the above-mentioned peptide (III), wherein X9 is P in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (ii) if said subject is the subject of (b), culturing CD8 T lymphocytes from a second subject comprising a C at a position corresponding to nucleotide 1057 in the nucleic acid sequence of human MTCH2 and/or a MTCH2 polypeptide comprising a proline residue at a position corresponding to residue 290 in the protein sequence of human MTCH2 in the presence of cells expressing a MHC class I molecule of the HLA-B*0801 allele loaded with the above-mentioned peptide (III), wherein X9 is A in said peptide, under conditions suitable for CD8 T lymphocyte expansion.
In an embodiment, the above-mentioned subject is an allogeneic stem cell transplantation (ASCT) recipient.
In another aspect, the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising: (a) determining whether a candidate donor expresses a MTCH2 nucleic acid comprising a C or a G at a position corresponding to nucleotide 1057 in the nucleic acid sequence of human MTCH2 (FIG. 3A, NCBI Reference Sequence: NM_014342.3), and/or a MTCH2 polypeptide comprising a proline or alanine residue at a position corresponding to residue 290 in the protein sequence of human MTCH2 (FIG. 3B, NCBI Reference Sequence: NP_055157.1) and (b)(i) if said candidate donor expresses a MTCH2 nucleic acid comprising a C at a position corresponding to nucleotide 1057 in the nucleic acid sequence of human MTCH2 and/or a MTCH2 polypeptide comprising a proline residue at a position corresponding to residue 290 in the protein sequence of human MTCH2, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-B*0801 allele loaded with the above-mentioned peptide (III), wherein X9 is A in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (b)(ii) if said candidate donor expresses a MTCH2 nucleic acid comprising a G at a position corresponding to nucleotide 1057 in the nucleic acid sequence of human MTCH2 and/or a MTCH2 polypeptide comprising an alanine residue at a position corresponding to residue 290 in the protein sequence of human MTCH2, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-B*0801 allele loaded with the above-mentioned peptide (III), wherein X9 is P in said peptide, under conditions suitable for CD8 T lymphocyte expansion.
In another aspect, the present invention provides a peptide of 50 amino acids or less comprising the sequence (IV)
Z1-X11X12VLKPGNX13X14-Z2 (IV)

wherein Z1 is an amino terminal modifying group or is absent; X11 is a sequence of 1 to 43 amino acids or is absent; X12 is S or T; X13 is an amino acid or is absent; X14 is a sequence of 1 to 43 amino acids or is absent; and Z2 is a carboxy terminal modifying group or is absent. In an embodiment, X11 is absent. In an embodiment, the above-mentioned X13 is an amino acid, in a further embodiment serine (S). In an embodiment, X14 is an amino acid, in a further embodiment a basic amino acid, more particularly lysine (K). In an embodiment, X12 is S. In another embodiment, X12 is T. In an embodiment, the above-mentioned peptide comprises the sequence SVLKPGNSK (SEQ ID NO:21). In a further embodiment, the peptide is SVLKPGNSK (SEQ ID NO:21). In another embodiment, the above-mentioned peptide comprises the sequence TVLKPGNSK (SEQ ID NO:22). In a further embodiment, the peptide is TVLKPGNSK (SEQ ID NO:22).
In another aspect, the present invention provides a method of treating cancer, said method comprising administering to a subject in need thereof an effective amount of CD8 T lymphocytes recognizing a MHC class I molecule of the HLA-A*0301 allele loaded with the above-mentioned peptide (IV).
In an embodiment, the above-mentioned method further comprises determining whether said subject expresses an ELF1 nucleic acid comprising an A or T at a position corresponding to nucleotide 1400 in the nucleic acid sequence of human ELF1 (FIGS. 4A and 4B, NCBI Reference Sequence: NM_172373.3) and/or an ELF1 polypeptide having a threonine or a serine at a position corresponding to residue 343 in the ELF1 protein sequence (FIG. 4C, NCBI Reference Sequence: NP_758961.1), wherein (a) if said subject expresses an ELF1 nucleic acid comprising an A at a position corresponding to nucleotide 1400 in the nucleic acid sequence of human ELF1 and/or an ELF1 polypeptide comprising a threonine residue at a position corresponding to residue 343 in the protein sequence of human ELF1, X12 is T in said peptide; (b) if said subject expresses an ELF1 nucleic acid comprising a T at a position corresponding to nucleotide 1400 in the nucleic acid sequence of human ELF1 and/or an ELF1 polypeptide comprising a serine residue at a position corresponding to residue 343 in the protein sequence of human ELF1, X12 is S in said peptide.
In an embodiment, the above-mentioned determining comprises sequencing a human ELF1 nucleic acid. In an embodiment, the above-mentioned CD8 T lymphocytes are in vitro expanded CD8 T lymphocytes.
In an embodiment, the above-mentioned method further comprises: (i) if said subject is the subject of (a), culturing CD8 T lymphocytes from a second subject comprising a T at a position corresponding to nucleotide 1400 in the nucleic acid sequence of human ELF1 and/or an ELF1 polypeptide comprising a serine residue at a position corresponding to residue 343 in the protein sequence of human ELF1 in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the peptide, wherein X12 is T in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (ii) if said subject is the subject of (b), culturing CD8 T lymphocytes from a second subject comprising an A at a position corresponding to nucleotide 1400 in the nucleic acid sequence of human ELF1 and/or an ELF1 polypeptide comprising a threonine residue at a position corresponding to residue 343 in the protein sequence of human ELF1 in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the peptide, wherein X12 is S in said peptide, under conditions suitable for CD8 T lymphocyte expansion.
In an embodiment, the above-mentioned subject is an allogeneic stem cell transplantation (ASCT) recipient.
In another aspect, the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising: (a) determining whether a candidate donor expresses an ELF1 nucleic acid comprising an A or T at a position corresponding to nucleotide 1400 in the nucleic acid sequence of human ELF1 (FIGS. 4A and 4B, NCBI Reference Sequence: NM_172373.3) and/or an ELF1 polypeptide having a threonine or a serine at a position corresponding to residue 343 in the ELF1 protein sequence (FIG. 4C, NCBI Reference Sequence: NP_758961.1) and (b)(i) if said candidate donor expresses an ELF1 nucleic acid comprising an A at a position corresponding to nucleotide 1400 in the nucleic acid sequence of human ELF1 and/or an ELF1 polypeptide having a threonine at a position corresponding to residue 343 in the ELF1 protein sequence, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the above-mentioned peptide (IV), wherein X12 is S in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (b)(ii) if said candidate donor expresses an ELF1 nucleic acid comprising a T at a position corresponding to nucleotide 1400 in the nucleic acid sequence of human ELF1 and/or an ELF1 polypeptide having a serine at a position corresponding to residue 343 in the ELF1 protein sequence, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the above-mentioned peptide (IV), wherein X12 is T in said peptide, under conditions suitable for CD8 T lymphocyte expansion.
In another aspect, the present invention provides a peptide of 50 amino acids or less comprising the sequence V
Z1-X15X16YDKGPFX17X18X19-Z2 (V)

wherein Z1 is an amino terminal modifying group or is absent; X15 is a sequence of 1 to 43 amino acids or is absent; X16 is an amino acid or is absent; X17 is R or W; X18 is an amino acid or is absent; X19 is a sequence of 1 to 43 amino acids or is absent; Z2 is a carboxy terminal modifying group or is absent. In an embodiment, the above-mentioned X16 is an amino acid, in a further embodiment a methionine (M). In an embodiment, X15 is an amino acid, in a further embodiment an alanine (A). In an embodiment, X18 is an amino acid, in a further embodiment a serine (S). In an embodiment, X19 is an amino acid, in a further embodiment a basic amino acid, more particularly lysine (K). In an embodiment, X17 is R. In another embodiment, X17 is W. In an embodiment, the above-mentioned peptide comprises the sequence AMYDKGPFRSK (SEQ ID NO:23). In an embodiment, the above-mentioned peptide is AMYDKGPFRSK (SEQ ID NO:23). In another embodiment, the above-mentioned peptide comprises the sequence AMYDKGPFWSK (SEQ ID NO:24). In an embodiment, the above-mentioned peptide is AMYDKGPFWSK (SEQ ID NO:24).
In another aspect, the present invention provides a method of treating cancer, said method comprising administering to a subject in need thereof an effective amount of CD8 T lymphocytes recognizing a MHC class I molecule of the HLA-A*0301 allele loaded with the above-mentioned peptide (V).
In an embodiment, the above-mentioned method further comprises determining whether said subject expresses an NQO1 nucleic acid comprising an C or T at a position corresponding to nucleotide 615 in the nucleic acid sequence of human NQO1 (FIG. 5A, NCBI Reference Sequence: NM_000903.2) and/or an NQO1 polypeptide having an arginine or a tryptophan at a position corresponding to residue 139 in the NQO1 protein sequence (FIG. 5B, NCBI Reference Sequence: NP_000894.1), wherein (a) if said subject expresses a NQO1 nucleic acid comprising an C at a position corresponding to nucleotide 615 in the nucleic acid sequence of human NQO1 and/or a NQO1 polypeptide comprising an arginine residue at a position corresponding to residue 139 in the protein sequence of human NQO1, X17 is R in said peptide; (b) if said subject expresses an NQO1 nucleic acid comprising a T at a position corresponding to nucleotide 615 in the nucleic acid sequence of human NQO1 and/or a NQO1 polypeptide comprising a tryptophan residue at a position corresponding to residue 139 in the protein sequence of human NQO1, X17 is W in said peptide.
In an embodiment, the above-mentioned determining comprises sequencing a human NQO1 nucleic acid. In an embodiment, the above-mentioned CD8 T lymphocytes are in vitro expanded CD8 T lymphocytes.
In an embodiment, the above-mentioned method further comprises: (i) if said subject is the subject of (a), culturing CD8 T lymphocytes from a second subject comprising a T at a position corresponding to nucleotide 615 in the nucleic acid sequence of human NQO1 and/or a NQO1 polypeptide comprising a tryptophan residue at a position corresponding to residue 139 in the protein sequence of human NQO1 in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the above-mentioned peptide (V), wherein X17 is R in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (ii) if said subject is the subject of (b), culturing CD8 T lymphocytes from a second subject comprising a C at a position corresponding to nucleotide 615 in the nucleic acid sequence of human NQO1 and/or a NQO1 polypeptide comprising an arginine residue at a position corresponding to residue 139 in the protein sequence of human NQO1 in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the above-mentioned peptide (V), wherein X17 is W in said peptide, under conditions suitable for CD8 T lymphocyte expansion.
In an embodiment, the above-mentioned subject is an allogeneic stem cell transplantation (ASCT) recipient.
In another aspect, the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising: (a) determining whether said subject expresses an NQO1 nucleic acid comprising an C or T at a position corresponding to nucleotide 615 in the nucleic acid sequence of human NQO1 (FIG. 5A, NCBI Reference Sequence: NM_000903.2) and/or an NQO1 polypeptide having an arginine or a tryptophan at a position corresponding to residue 139 in the NQO1 protein sequence (FIG. 5B, NCBI Reference Sequence: NP_000894.1) and (b)(i) if said candidate donor expresses a NQO1 nucleic acid comprising an C at a position corresponding to nucleotide 615 in the nucleic acid sequence of human NQO1 and/or a NQO1 polypeptide comprising an arginine residue at a position corresponding to residue 139 in the protein sequence of human NQO1, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the above-mentioned peptide (V), wherein X17 is W in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (b)(ii) if said candidate donor expresses a NQO1 nucleic acid comprising a T at a position corresponding to nucleotide 615 in the nucleic acid sequence of human NQO1 and/or a NQO1 polypeptide comprising a tryptophan residue at a position corresponding to residue 139 in the protein sequence of human NQO1, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the above-mentioned peptide (V), wherein X17 is R in said peptide, under conditions suitable for CD8 T lymphocyte expansion.
In another aspect, the present invention provides a peptide of 50 amino acids or less comprising the sequence VI
Z1-X20X21X22SLPTSPX23X24-Z2 (VI)

wherein Z1 is an amino terminal modifying group or is absent; X20 is a sequence of 1 to 43 amino acids or is absent; X21 is an amino acid or is absent; X22 is an amino acid or is absent; X23 is G or R; X24 is a sequence of 1 to 43 amino acids or is absent; Z2 is a carboxy terminal modifying group or is absent. In an embodiment, the peptide has a length of 8 to 12 amino acids. In an embodiment, X22 is an amino acid, in a further embodiment valine (V). In an embodiment, X21 is an amino acid, in a further embodiment arginine (R). In an embodiment, X23 is glycine (G), in another embodiment X23 is arginine (R). In an embodiment, the above-mentioned peptide comprises the sequence RVSLPTSPG (SEQ ID NO:25). In an embodiment, the above-mentioned peptide is RVSLPTSPG (SEQ ID NO:25). In another embodiment, the above-mentioned peptide comprises the sequence RVSLPTSPR (SEQ ID NO:26). In an embodiment, the above-mentioned peptide is RVSLPTSPR (SEQ ID NO:26).
In another aspect, the present invention provides a method of treating cancer, said method comprising administering to a subject in need thereof an effective amount of CD8 T lymphocytes recognizing a MHC class I molecule of the HLA-A*0301 allele loaded with the above-mentioned peptide of sequence VI.
In an embodiment, the above-mentioned method further comprising determining whether said subject expresses a KIAA0226L nucleic acid comprising a G or A at a position corresponding to nucleotide 1059 in the nucleic acid sequence of human KIAA0226L (FIGS. 6A and 6B, NCBI Reference Sequence: NM_025113.2) and/or an KIAA0226L polypeptide having a glycine or an arginine at a position corresponding to residue 152 in the KIAA0226L protein sequence (FIG. 6C, NCBI Reference Sequence: NP_079389.2), wherein (a) if said subject expresses a KIAA0226L nucleic acid comprising a G at a position corresponding to nucleotide 1059 in the nucleic acid sequence of human KIAA0226L and/or a KIAA0226L polypeptide comprising a glycine residue at a position corresponding to residue 152 in the protein sequence of human KIAA0226L, X23 is G in said peptide of sequence VI; (b) if said subject expresses a KIAA0226L nucleic acid comprising an A at a position corresponding to nucleotide 1059 in the nucleic acid sequence of human a KIAA0226L and/or a KIAA0226L polypeptide comprising an arginine residue at a position corresponding to residue 152 in the protein sequence of human KIAA0226L, X23 is R in said peptide of sequence VI.
In another embodiment, the determining comprises sequencing a human KIAA0226L nucleic acid. In an embodiment, the above-mentioned CD8 T lymphocytes are in vitro expanded CD8 T lymphocytes.
In an embodiment, the above-mentioned method further comprises: (i) if said subject is the subject of (a) above, culturing CD8 T lymphocytes from a second subject comprising an A at a position corresponding to nucleotide to nucleotide 1059 in the nucleic acid sequence of human KIAA0226L and/or a KIAA0226L polypeptide comprising an arginine residue at a position corresponding to residue 152 in the protein sequence of human KIAA0226L in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the peptide of sequence VI, wherein X23 is G in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (ii) if said subject is the subject of (b) above, culturing CD8 T lymphocytes from a second subject comprising a G at a position corresponding to nucleotide 1059 in the nucleic acid sequence of human KIAA0226L and/or a KIAA0226L polypeptide comprising a glycine residue at a position corresponding to residue 152 in the protein sequence of human KIAA0226L in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the peptide of sequence VI, wherein X23 is R in said peptide, under conditions suitable for CD8 T lymphocyte expansion.
In an embodiment, the above-mentioned subject is an allogeneic stem cell transplantation (ASCT) recipient.
In another aspect, the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising: (a) determining whether a candidate donor expresses a KIAA0226L nucleic acid comprising a G or A at a position corresponding to nucleotide 1059 in the nucleic acid sequence of human KIAA0226L (FIGS. 6A and 6B, NCBI Reference Sequence: NM_025113.2) and/or an KIAA0226L polypeptide having a glycine or an arginine at a position corresponding to residue 152 in the KIAA0226L protein sequence (FIG. 6C, NCBI Reference Sequence: NP_079389.2) and (b)(i) if said candidate donor expresses a KIAA0226L nucleic acid comprising G at a position corresponding to nucleotide 1059 in the nucleic acid sequence of human KIAA0226L and/or a KIAA0226L polypeptide comprising a glycine residue at a position corresponding to residue 152 in the protein sequence of human KIAA0226L, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the peptide of sequence VI, wherein X23 is R in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (b)(ii) if said subject expresses a KIAA0226L nucleic acid comprising an A at a position corresponding to nucleotide 1059 in the nucleic acid sequence of human a KIAA0226L and/or a KIAA0226L polypeptide comprising an arginine residue at a position corresponding to residue 152 in the protein sequence of human KIAA0226L, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the peptide of sequence VI, wherein X23 is G in said peptide, under conditions suitable for CD8 T lymphocyte expansion.
In another aspect, the present invention provides a peptide of 50 amino acids or less comprising the sequence VII
Z1-X25X26X27GNPGTFX28X29-Z2 (VII)
wherein Z1 is an amino terminal modifying group or is absent; X25 is a sequence of 1 to 43 amino acids or is absent; X26 is an amino acid or is absent; X27 is an amino acid or is absent; X28 is K or N; X29 is a sequence of 1 to 43 amino acids or is absent; Z2 is a carboxy terminal modifying group or is absent. In an embodiment, the peptide has a length of 8 to 12 amino acids. In an embodiment, X27 is an amino acid, more specifically methionine (M). In an embodiment, X26 is an amino acid, more specifically valine (V). In an embodiment, X28 is K. In another embodiment, X28 is N. In an embodiment, the peptide comprises the sequence VMGNPGTFK (SEQ ID NO: 27). In a further embodiment, the peptide is VMGNPGTFK (SEQ ID NO: 27). In another embodiment, the peptide comprises the sequence VMGNPGTFN (SEQ ID NO: 28). In an embodiment, the peptide is VMGNPGTFN (SEQ ID NO: 28).
In another aspect, the present invention provides a method of treating cancer, said method comprising administering to a subject in need thereof an effective amount of CD8 T lymphocytes recognizing a MHC class I molecule of the HLA-A*0301 allele loaded with the above-mentioned peptide of sequence VII.
In an embodiment, the above-mentioned method further comprises determining whether said subject expresses an RMDN1 nucleic acid comprising an A or C at a position corresponding to nucleotide 316 in the nucleic acid sequence of human RMDN1 (FIG. 7A, NCBI Reference Sequence: NM_016033.2) and/or an RMDN1 polypeptide having a lysine or an asparagine at a position corresponding to residue 52 in the RMDN1 protein sequence (FIG. 7B, NCBI Reference Sequence: NP_057117.2), wherein (a) if said subject expresses an RMDN1 nucleic acid comprising an A at a position corresponding to nucleotide 316 in the nucleic acid sequence of human RMDN1 and/or an RMDN1 polypeptide comprising a lysine residue at a position corresponding to residue 52 in the protein sequence of human RMDN1, X28 is K in said peptide of sequence VII; (b) if said subject expresses an RMDN1 nucleic acid comprising a C at a position corresponding to nucleotide 316 in the nucleic acid sequence of human RMDN1 and/or a RMDN1 polypeptide comprising an asparagine residue at a position corresponding to residue 52 in the protein sequence of human RMDN1, X28 is N in said peptide of sequence VII.
In another embodiment, the determining comprises sequencing a human RMDN1 nucleic acid. In an embodiment, the above-mentioned CD8 T lymphocytes are in vitro expanded CD8 T lymphocytes.
In an embodiment, the above-mentioned method further comprises: (i) if said subject is the subject of (a), culturing CD8 T lymphocytes from a second subject comprising a C at a position corresponding to nucleotide 316 in the nucleic acid sequence of human RMDN1 and/or a RMDN1 polypeptide comprising an asparagine residue at a position corresponding to residue 52 in the protein sequence of human RMDN1 in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the peptide of sequence VII, wherein X28 is K in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (ii) if said subject is the subject of (b) above, culturing CD8 T lymphocytes from a second subject comprising an A at a position corresponding to nucleotide 316 in the nucleic acid sequence of human RMDN1 and/or a RMDN1 polypeptide comprising a lysine residue at a position corresponding to residue 52 in the protein sequence of human RMDN1 in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the peptide of sequence VII, wherein X28 is N in said peptide, under conditions suitable for CD8 T lymphocyte expansion.
In an embodiment, the above-mentioned subject is an allogeneic stem cell transplantation (ASCT) recipient.
In another aspect, the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising: (a) determining whether a candidate donor expresses an RMDN1 nucleic acid comprising an A or C at a position corresponding to nucleotide 316 in the nucleic acid sequence of human RMDN1 (FIG. 7A, NCBI Reference Sequence: NM_016033.2) and/or an RMDN1 polypeptide having a lysine or an asparagine at a position corresponding to residue 52 in the RMDN1 protein sequence (FIG. 7B, NCBI Reference Sequence: NP_057117.2) and (b)(i) if said subject expresses an RMDN1 nucleic acid comprising an A at a position corresponding to nucleotide 316 in the nucleic acid sequence of human RMDN1 and/or an RMDN1 polypeptide comprising a lysine residue at a position corresponding to residue 52 in the protein sequence of human RMDN1, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the peptide of sequence VII, wherein X28 is N in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (b)(ii) if said subject expresses an RMDN1 nucleic acid comprising a C at a position corresponding to nucleotide 316 in the nucleic acid sequence of human RMDN1 and/or a RMDN1 polypeptide comprising an asparagine residue at a position corresponding to residue 52 in the protein sequence of human RMDN1, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the peptide of sequence VII, wherein X28 is R in said peptide, under conditions suitable for CD8 T lymphocyte expansion.
In an embodiment, Z1 is absent in the above-mentioned peptide (I)-(VII). In an embodiment, Z2 is absent in the above-mentioned peptide (I)-(VII).
In another aspect, the present invention provides a MiHA identified by the above-mentioned method. In an embodiment, the MiHA is a peptide of sequences (I)-(VII) as defined herein.
In another aspect, the present invention provides a nucleic acid encoding the above-mentioned peptide (I)-(VII).
In another aspect, the present invention provides an isolated major histocompatibility complex (MHC) class I molecule loaded with the peptide (I)-(VII). In another aspect, the present invention provides an isolated cell expressing at its surface a MHC class I molecule loaded with the above-mentioned peptide (I)-(VII).
In an embodiment, the major histocompatibility complex (MHC) class I molecule is of the HLA-A*0301, HLA-B*0801 or HLA-B*4403 allele. In a further embodiment, the major histocompatibility complex (MHC) class I molecule is of the HLA-A*0301 allele. In another embodiment, the major histocompatibility complex (MHC) class I molecule is of the HLA-B*0801 allele. In another embodiment, the major histocompatibility complex (MHC) class I molecule is of the HLA-B*4403 allele.
Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
BRIEF DESCRIPTION OF DRAWINGS
In the appended drawings:
FIGS. 1A to C show the nucleotide sequence of human centromer protein F, 350/400 kDa (mitosin) (CENPF) cDNA (SEQ ID NO:1). The coding region is in italics;
FIG. 1D shows the amino acid sequence of human CENPF polypeptide (SEQ ID NO:2);
FIG. 2A shows the nucleotide sequence of human ZW10 interactor (ZWINT) cDNA (SEQ ID NO:3). The coding region is in italics;
FIG. 2B shows the amino acid sequence of human ZWINT polypeptide (SEQ ID NO:4);
FIG. 3A shows the nucleotide sequence of human mitochondrial carrier homolog 2 (MTCH2) cDNA (SEQ ID NO:5). The coding region is in italics;
FIG. 3B shows the amino acid sequence of human MTCH2 polypeptide (SEQ ID NO:6);
FIGS. 4A and 4B show the nucleotide sequence of human ELF1 [E74-like factor 1 (ets domain transcription factor)] cDNA (SEQ ID NO:7). The coding region is in italics;
FIG. 4C shows the amino acid sequence of human ELF1 polypeptide (SEQ ID NO:8);
FIGS. 5A and 5B show the nucleotide sequence of human NQO1 [NAD(P)H dehydrogenase, quinone 1] cDNA (SEQ ID NO:9). The coding region is in italics;
FIG. 5C shows the amino acid sequence of human NQO1 polypeptide (SEQ ID NO:10);
FIGS. 6A and 6B show the nucleotide sequence of human KIAA0226L cDNA (SEQ ID NO:11). The coding region is in italics;
FIG. 6C shows the amino acid sequence of human KIAA0226L polypeptide (SEQ ID NO:12);
FIG. 7A shows the nucleotide sequence of human RMDN1 cDNA (SEQ ID NO:13). The coding region is in italics; and
FIG. 7B shows the amino acid sequence of human RMDN1 polypeptide (SEQ ID NO:14).
 DISCLOSURE OF INVENTION
Terms and symbols of genetics, molecular biology, biochemistry and nucleic acid used herein follow those of standard treatises and texts in the field, e.g. Kornberg and Baker, DNA Replication, Second Edition (W.H. Freeman, New York, 1992); Lehninger, Biochemistry, Second Edition (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics, Second Edition (Wiley-Liss, New York, 1999); Eckstein, editor, Oligonucleotides and Analogs: A Practical Approach (Oxford University Press, New York, 1991); Gait, editor, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1984); and the like. All terms are to be understood with their typical meanings established in the relevant art.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.
Described herein is a novel method for human MiHA discovery, novel MiHAs identified using this method, as well as uses of the novel MiHAs. One of the features of the method is the inclusion of personalized translated transcriptome and/or exome in the database used for peptide identification by mass spectroscopy (MS). Candidate MiHAs are identified by comparing the personalized transcriptome and/or exome to a reference genome and/or to the transcriptome and/or exome of an HLA-matched subject (e.g., an HLA-identical sibling).
Accordingly, in a first aspect, the present invention provides a method of identifying a minor histocompatibility antigen (MiHA) candidate, the method comprising: (a) isolating and determining the sequence of MHC-associated peptides (MAPs) in a first cell sample from a first subject; (b) performing a whole transcriptome and/or exome sequencing on a second cell sample obtained from said first subject; (c) comparing the sequenced whole transcriptome and/or exome to a reference genome to identify single nucleotide variations (SNVs) between the transcriptome and/or exome of said first subject and the reference genome; (d) in silico translating the sequences containing the identified SNVs to identify peptide sequences comprising at least one non-synonymous mutation caused by said SNVs; (e) comparing the sequences of the MAPs isolated in (a) with the peptide sequences identified in (d); and (f) identifying a MiHA candidate based on said comparison.
In an embodiment, the MiHA candidate is a MAP whose sequence comprises at least one mutation relative to the corresponding sequence translated from the reference genome.
In another aspect, the present invention provides a method of identifying a minor histocompatibility antigen (MiHA) candidate, the method comprising: (a) isolating MHC-associated peptides (MAPs) in a first cell sample from a first and second subjects, wherein said first and second subjects are human leukocyte antigen (HLA)-matched; (b) performing a whole transcriptome and/or exome sequencing on a second cell sample obtained from said first and second subjects; (c) comparing the sequenced whole transcriptomes and/or exomes to identify single nucleotide variations (SNVs) between the transcriptomes and/or exomes of said first and second subjects; (d) in silico translating the sequences containing the identified SNVs to identify peptide sequences comprising at least one non-synonymous mutation caused by said SNVs; (e) comparing the sequences of the MAPs isolated in (a) with the peptide sequences identified in (d); and (f) identifying a MiHA candidate based on said comparison. In an embodiment, the MiHA candidate is a MAP present in the first cell sample from said first subject but absent from the first cell sample from said second subject.
The term “reference genome” as used herein refers to the human genome assemblies reported in the literature, and includes for example the Genome Reference Consortium Human Build 37 (GRCh37, Genome Reference Consortium; The International Human Genome Sequencing Consortium. Nature. 2004; 431:931-945), Hs_Celera_WGSA (Celera Genomics; Istrail S. et al., Proc Natl Acad Sci USA. 2004 Feb. 17; 101(7):1916-21). Epub 2004 Feb. 9), HuRef and HuRefPrime (J. Craig Venter Institute; Levy S, et al. PLoS Biology. 2007; 5:2113-2144), YH1 and BGIAF (Beijing Genomics Institute; Li R, et al. Genome Research. 2010; 20: 265-272), as well as HsapALLPATHS1 (Broad Institute). In an embodiment, the reference genome is GRCh37.
In various embodiments, the above-noted first sample may be from any source that contains cells expressing MHC class I molecules, including a tissue or body fluid from the subject, such as blood, serum, immune cells (e.g., lymphocytes), blood cells (e.g., PBMCs or a subset thereof), tissues, or a cell line derived from primary cells. In an embodiment, the first sample is a blood cell sample, for example a PBMC sample, or a cell line derived from blood cells such as PBMCs (e.g., an immortalized cell line). Methods for generating a cell line from primary cells, or for immortalizing primary cells, are known in the art and include, for example, immortalization of primary cells by recombinant expression of human telomerase reverse transcriptase (TERT) (Barsov E V, Curr Protoc Immunol. 2011 November; Chapter 7: Unit 7.21B), immortalization by recombinant expression of viral genes such as Simian virus 40 (SV40) T antigen, adenovirus E1A and E1B, human papillomavirus (HPV) E6 and E7 and Epstein-Barr Virus (EBV), as well as inactivation of tumor suppression genes such as p53 or Rb. Methods for immortalization of B lymphocytes by EBV are disclosed in Tosato G and Cohen J I. Curr Protoc Immunol. 2007 February; Chapter 7: Unit 7.22. Products/reagents for immortalizing mammalian cells are commercially available, for example from ATCC™. In an embodiment, the first sample is an immortalized cell line derived from primary cells obtained from the subject, in a further embodiment an immortalized B cell line, such as an EBV-transformed B lymphoblastoid cell line (B-LCL).
Methods for isolating MHC-associated peptides (MAPs) from a cell sample are well known in the art. The most commonly used technique is mild acid elution (MAE) of MHC-associated peptides from living cells, as described in Fortier et al. (J. Exp. Med. 205(3): 595-610, 2008). Another technique is immunoprecipitation or affinity purification of peptide-MHC class I complexes followed by peptide elution (see, e.g., Gebreselassie et al., Hum Immunol. 2006 November; 67(11): 894-906). Two high-throughput strategies based on the latter approach have been implemented. The first is based on transfection of cell lines with expression vectors coding soluble secreted MHCs (lacking a functional transmembrane domain) and elution of peptides associated with secreted MHCs (Barnea et al., Eur J Immunol. 2002 January; 32(1):213-22; and Hickman H D et al., J Immunol. 2004 Mar. 1; 172(5):2944-52). The second approach hinges on chemical or metabolic labeling to provide quantitative profiles of MHC-associated peptides (Weinzierl A O et al., Mol Cell Proteomics. 2007 January; 6(1):102-13. Epub 2006 Oct. 29; Lemmel C et al., Nat Biotechnol. 2004 April; 22(4):450-4. Epub 2004 Mar. 7; Milner E, Mol Cell Proteomics. 2006 February; 5(2):357-65. Epub 2005 Nov. 4).
Eluted MAPs may be subjected to any purification/enrichment steps, including size exclusion chromatography or ultrafiltration (using a filter with a cut-off of about 5000 Da, for example about 3000 Da), and/or ion exchange chromatography (e.g., cation exchange chromatography), prior to further analysis. The sequence of the eluted MAPs may be determined using any method known in the art for sequencing peptides/proteins, such as mass spectroscopy (as described below) and the Edman degradation reaction.
In various embodiments, the above-noted second sample may be from any source that contains genomic DNA, RNA, and/or proteins, for example a tissue or body fluid from the subject, such as blood, serum, immune cells (e.g., lymphocytes), blood cells (e.g., PBMCs), tissues, or a cell line derived from primary cells (as described above). In an embodiment, the second sample is an immortalized cell line derived from primary cells obtained from the subject, in a further embodiment an immortalized B cell line, such as an EBV-transformed B lymphoblastoid cell line (B-LCL). The cell sample may be subjected to commonly used isolation and/or purification techniques for enrichment in nucleic acids (genomic DNA, mRNA) and/or proteins.
In an embodiment, transcriptome libraries are generated/constructed from the RNA obtained from the sample. Transcriptome library construction may include one or more of the following steps: poly-A mRNA enrichment/purification; RNA fragmentation and priming for cDNA synthesis; reverse transcription (RT) (using random primers); second round of RT to generate a double-stranded cDNA, cDNA purification; end repair of fragmented cDNA, adenylation of the 3′ ends, ligation of adaptors and enrichment of DNA fragments containing adapter molecules. Kits suitable for transcriptome library construction are commercially available, for example from Life Technologies (Ambion® RNA-Seq Library Construction Kit), Applied Biosystems (AB Library Builder™ Whole Transcriptome Core Kit), Qiagen (Quantilect™ Whole Transcriptome Kit) and Sigma-Aldrich (TransPlex® Complete Whole Transcriptome Amplification Kit)
In an embodiment, genomic libraries are generated/constructed from the genomic DNA obtained from the sample. Genomic library construction may include one or more of the following steps: DNA shearing, DNA end repair, 3′ ends adenylation, ligation of adaptors, purification of ligation products and amplification (e.g., PCR) to enrich DNA fragments that have adapter molecules. Kits suitable for genomic library construction are commercially available, for example from Illumina (TruSeq™ DNA Sample Preparation Kit (v2) (Cat. No. FC-930-1021), Life Technologies (SOLiD® Fragment Library Construction Kit and New England BioLabs (NEBNext® DNA Library Preparation).
In another embodiment, the genomic (DNA-Seq) libraries are subjected to an enrichment step to sequence only the coding portion (exome) of the human genome. Kits suitable for exome enrichment are commercially available, for example from Illumina (TruSeq™ exome enrichment kit, FC-930-1012), Life Technologies (TargetSeq™ Exome and Custom Enrichment System, A14060-A14063), FlexGen (FleXome whole exome enrichment kit v2), Roche NimbleGen (SeqCap EZ Human Exome Library v2.0) and Agilent Technologies (SureSelect All Exon kits)
Methods to perform whole transcriptome or exome sequencing (RNA-Seq) are known in the art (see, for example, Wang et al., Nature Reviews Genetics 10, 57-63, January 2009; Genome Biology 2011, 12(9), Exome sequencing special issue). Various platforms for performing whole transcriptome/exome sequencing exist, such as the Illumina Genome Analyzer platform, the Applied Biosystems (ABI) Solid™ Sequencing platform or Life Science's 454 Sequencing platform (Roche).
The identification of single nucleotide variations (SNVs) or single nucleotide polymorphisms (SNPs) between two or more sequences, for example between (i) the transcriptome and/or exome of a subject and a reference genome and/or (ii) the transcriptomes and/or exomes of two different subjects, may be performed using any sequence comparison/SNP identification methods or tools, including the SNP calling program Casava™ from Illumina, SNPdetector (Zhang et al., PLoS Comput Biol. 2005 October; 1(5): e53), the SNP & Variation Suite from Golden Helix, the Genome-wide human SNP arrays from Affymetrix, the SAMtools mpileup from MassGenomics, etc.
The in silico translation of nucleic acid sequences to protein sequences may be performed using any suitable softwares or tools, including the ExPASy Translate tool, Vector NTI™ (Life Technologies), pyGeno (Granados et al., 2012), Virtual Ribosome (CBS, University of Denmark), etc. The in silico translation of transcriptomes and/or exomes permits the identification of peptide sequences comprising at least one non-synonymous mutation caused by the SNVs. In an embodiment, all possible amino acid (aa) sequence variants of 15 amino acids or less (in embodiments, 14, 13, 12, 11 amino acids or less) comprising at least one non-synonymous mutation are computed, listed and used in the comparing step (e). Thus, for each non-synonymous mutation caused by the SNVs, a window of 90 bp (84, 78, 72 or 66 bp) around each one of the polymorphic positions is computed to obtain the list of every possible amino acid (aa) sequence variant defined by these 90 bp (84, 78, 72 or 66 bp) (30, 28, 26, 24 or 22 aa) windows. In this way, a list of most possible aa sequences of at most 15 aa (in embodiments, 14, 13, 12, 11 aa) affected by non-synonymous polymorphisms may be obtained. For the identification of MHC class II-associated MAPs (which may be longer, up to about 30 amino acids), all possible amino acid (aa) sequence variants of for example 30 amino acids or less (in embodiments, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12 amino acids or less) comprising at least one non-synonymous mutation are computed, listed and used in the comparing step (e). Thus, for each non-synonymous mutation caused by the SNVs, a window of 180 bp around each one of the polymorphic positions is computed to obtain the list of every possible amino acid (aa) sequence variant defined by these 180 bp (60 aa) windows. In this way, a list of most possible aa sequences of at most 30 aa affected by non-synonymous polymorphisms may be obtained.
In an embodiment, all possible amino acid (aa) sequence variants of 12 or 11 amino acids or less comprising at least one non-synonymous mutation are computed, listed and used in the comparing step (e). Thus, for each non-synonymous mutation caused by the SNVs, a window of 72 or 66 bp around each one of the polymorphic positions is computed to obtain the list of every possible amino acid (aa) sequence variant defined by these 72 or 66 bp (24 or 22 aa) windows. In this way, a list of most possible aa sequences of at most 12 or 11 aa affected by non-synonymous polymorphisms was obtained.
In embodiment, the above-noted peptide sequences have a length of about 7 to about 15 amino acids (e.g., 7, 8, 9, 10, 11, 12, 13, 14 or 15), in a further embodiment of about 8 or 9 to about 11 or 12 amino acids (e.g., 8, 9, 10, 11 or 12).
In an embodiment, the comparison of the sequences of the MAPs isolated from the first sample with the peptide sequences derived from the transcriptome and/or exome identified above (i.e. comprising at least one non-synonymous mutation caused by the SNVs) comprises subjecting the isolated MAPs to mass spectrometry and comparing the MS spectra obtained with the peptide sequences derived from the transcriptome and/or exome. In an embodiment, the mass spectrometry is liquid chromatography-mass spectrometry (LC-MS), in a further embodiment LC-MS coupled to peptide mass fingerprinting (LC-MS/MS).
In an embodiment, the method further comprises determining the binding of the MiHA candidate identified to a MHC class I molecule. The binding may be a predicted binding affinity (IC50) of peptides to the allelic products, which may be obtained using tools such as the NetMHCcons software version 1.0 (http://www.cbs.dtu.dk/services/NetMHCcons/) (Karosiene et al., 2011). An overview of the various available MHC class I peptide binding tools is provided in Peters B et al., PLoS Comput Biol 2006, 2(6):e65; Trost et al., Immunome Res 2007, 3(1):5; Lin et al., BMC Immunology 2008, 9:8)
In an embodiment, peptides with a predicted IC50 below 50 nM are considered as strong binders and peptides with an IC50 between about 50 and about 500 nM are considered as weak binders.
The binding of the MiHA candidate identified to a MHC class I molecule may be determined using other known methods, for example the 12 Peptide Binding Assay. 12 cell lines are deficient in TAP but still express low amounts of MHC class I on the surface of the cells. The T2 binding assay is based upon the ability of peptides to stabilize the MHC class I complex on the surface of the T2 cell line. T2 cells are incubated with a specific peptide (e.g., a candidate MiHA), stabilized MHC class I complexes are detected using a pan-HLA class I antibody, an analysis is carried out (by flow cytometry, for example) and binding is assessed in relation to a non-binding negative control. The presence of stabilized peptide/MHC class I complexes at the surface is indicative that the peptide (e.g., candidate MiHA) binds to MHC class I molecules.
The binding of a peptide of interest (e.g., candidate MiHA) to MHC may also be assessed based on its ability to inhibit the binding of a radiolabeled probe peptide to MHC molecules. MHC molecules are solubilized with detergents and purified by affinity chromatography. They are then incubated for 2 days at room temperature with the inhibitor peptide (e.g., candidate MiHA) and an excess of a radiolabeled probe peptide, in the presence of a cocktail of protease inhibitors. At the end of the incubation period, MHC-peptide complexes are separated from unbound radiolabeled peptide by size-exclusion gel-filtration chromatography, and the percent bound radioactivity is determined. The binding affinity of a particular peptide for an MHC molecule may be determined by co-incubation of various doses of unlabeled competitor peptide with the MHC molecules and labeled probe peptide. The concentration of unlabeled peptide required to inhibit the binding of the labeled peptide by 50% (IC50) can be determined by plotting dose versus % inhibition (see, e.g., Current Protocols in Immunology (1998) 18.3.1-18.3.19, John Wiley & Sons, Inc.).
The binding of the MiHA candidate identified to a MHC class I molecule may be determined using an epitope discovery system, such as the ProImmune REVEAL & ProVE® epitope discovery system.
In another aspect, the present invention provides a peptide (e.g., an isolated or synthetic peptide) of 50 amino acids or less comprising the sequence (I) to (VII) described herein.
In another aspect, the present invention provides a peptide (e.g., an isolated or synthetic peptide) of 50 amino acids or less comprising the sequence (I):
Z1-X1LQEKFX2SX3-Z2
wherein Z1 is an amino terminal modifying group or is absent; X1 is a sequence of 1 to 43 amino acids or is absent; X2 is L or S; X3 is a sequence of 1 to 43 amino acids or is absent; and Z2 is a carboxy terminal modifying group or is absent.
In another aspect, the present invention provides a peptide (e.g., an isolated or synthetic peptide) of 50 amino acids or less comprising the sequence (II):
Z1-X4ELDX5VFQX6X7-Z2 (II)
wherein Z1 is an amino terminal modifying group or is absent; X4 is a sequence of 1 to 43 amino acids or is absent; X5 is G or R; X6 is an amino acid or is absent; X7 is a sequence of 1 to 43 amino acids or is absent; and Z2 is a carboxy terminal modifying group or is absent.
In another aspect, the present invention provides a peptide (e.g., an isolated or synthetic peptide) of 50 amino acids or less comprising the sequence (III):
Z1-X8LFFRKVX9X10-Z2 (III)
wherein Z1 is an amino terminal modifying group or is absent; X8 is a sequence of 1 to 43 amino acids or is absent; X9 is P or A; X10 is a sequence of 1 to 43 amino acids or is absent; and Z2 is a carboxy terminal modifying group or is absent.
In another aspect, the present invention provides a peptide (e.g., an isolated or synthetic peptide) of 50 amino acids or less comprising the sequence (IV):
Z1-X11X12VLKPGNX13X14-Z2 (IV)
wherein Z1 is an amino terminal modifying group or is absent; X11 is a sequence of 1 to 43 amino acids or is absent; X12 is S or T; X13 is an amino acid or is absent; X14 is a sequence of 1 to 43 amino acids or is absent; and Z2 is a carboxy terminal modifying group or is absent.
In another aspect, the present invention provides a peptide (e.g., an isolated or synthetic peptide) of 50 amino acids or less comprising the sequence (V):
Z1-X15X16YDKGPFX17X18X19-Z2 (V)
wherein Z1 is an amino terminal modifying group or is absent; X15 is a sequence of 1 to 43 amino acids or is absent; X16 is an amino acid or is absent; X17 is R or W; X18 is an amino acid or is absent; X19 is a sequence of 1 to 43 amino acids or is absent; Z2 is a carboxy terminal modifying group or is absent.
In another aspect, the present invention provides a peptide (e.g., an isolated or synthetic peptide) of 50 amino acids or less comprising the sequence (VI):
Z1-X20X21X22SLPTSPX23X24-Z2 (VI)
wherein Z1 is an amino terminal modifying group or is absent; X20 is a sequence of 1 to 43 amino acids or is absent; X21 is an amino acid or is absent; X22 is an amino acid or is absent; X23 is G or R; X24 is a sequence of 1 to 43 amino acids or is absent; Z2 is a carboxy terminal modifying group or is absent.
In another aspect, the present invention provides a peptide (e.g., an isolated or synthetic peptide) of 50 amino acids or less comprising the sequence (VII):
Z1-X25X26X27GNPGTFX28X29-Z2 (VII)
wherein Z1 is an amino terminal modifying group or is absent; X25 is a sequence of 1 to 43 amino acids or is absent; X26 is an amino acid or is absent; X27 is an amino acid or is absent; X28 is K or N; X29 is a sequence of 1 to 43 amino acids or is absent; Z2 is a carboxy terminal modifying group or is absent.
In another aspect, the present invention provides a peptide of 50 amino acids or less comprising any one of the sequences (I) to (VII) as defined herein.
In general, peptides presented in the context of HLA class I vary in length from about 7 to about 15 amino acid residues, and a longer peptide (e.g., of 50 amino acids or less) can be enzymatically processed to a peptide of such length. In embodiments, the peptide is 45, 40, 35, 30, 25, 20 or 15 amino acids or less. A peptide comprising the above-noted sequence/motif provided by the invention typically is at least 7 amino acids in length but preferably at least 8 or 9 amino acids. The upper length of a peptide provided by the invention is no more than 15 amino acids, but preferably no more than about 13, 12 or 11 amino acids in length. In embodiments, the above-mentioned peptide is about 8 to 12 amino acids long (e.g., 8, 9, 10, 11 or 12 amino acids long), small enough for a direct fit in an HLA class I molecule, but it may also be larger, between 12 to about 20, 25, 30, 35, 40, 45 or 50 amino acids and presented by HLA molecules only after cellular uptake and intracellular processing by the proteasome and transport before presentation in the groove of an MHC molecule.
The term “amino acid” as used herein includes both L- and D-isomers of the naturally occurring amino acids as well as other amino acids (e.g., naturally-occurring amino acids, non-naturally-occurring amino acids, amino acids which are not encoded by nucleic acid sequences, etc.) used in peptide chemistry to prepare synthetic analogs of peptides. Examples of naturally occurring amino acids are glycine, alanine, valine, leucine, isoleucine, serine, threonine, etc.
Other amino acids include for example non-genetically encoded forms of amino acids, as well as a conservative substitution of an L-amino acid. Naturally-occurring non-genetically encoded amino acids include, for example, beta-alanine, 3-amino-propionic acid, 2,3-diaminopropionic acid, alpha-aminoisobutyric acid (Aib), 4-amino-butyric acid, N-methylglycine (sarcosine), hydroxyproline, ornithine (e.g., L-ornithine), citrulline, t-butylalanine, t-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine, norleucine (Nle), norvaline, 2-naphthylalanine, pyridylalanine, 3-benzothienyl alanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1,2,3,4-tetrahydro-isoquinoline-3-carboxylix acid, beta-2-thienylalanine, methionine sulfoxide, L-homoarginine (Hoarg), N-acetyl lysine, 2-amino butyric acid, 2-amino butyric acid, 2,4,-diaminobutyric acid (D- or L-), p-aminophenylalanine, N-methylvaline, homocysteine, homoserine (HoSer), cysteic acid, epsilon-amino hexanoic acid, delta-amino valeric acid, or 2,3-diaminobutyric acid (D- or L-), etc. These amino acids are well known in the art of biochemistry/peptide chemistry.
In embodiments, the peptides of the present invention include peptides with altered sequences containing substitutions of functionally equivalent amino acid residues, relative to the above-mentioned sequences. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity (having similar physico-chemical properties) which acts as a functional equivalent, resulting in a silent alteration. Substitution for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, positively charged (basic) amino acids include arginine, lysine and histidine (as well as homoarginine and ornithine). Nonpolar (hydrophobic) amino acids include leucine, isoleucine, alanine, phenylalanine, valine, proline, tryptophan and methionine. Uncharged polar amino acids include serine, threonine, cysteine, tyrosine, asparagine and glutamine. Negatively charged (acidic) amino acids include glutamic acid and aspartic acid. The amino acid glycine may be included in either the nonpolar amino acid family or the uncharged (neutral) polar amino acid family. Substitutions made within a family of amino acids are generally understood to be conservative substitutions.
The above-mentioned peptide may comprise all L-amino acids, all D-amino acids or a mixture of L- and D-amino acids. In an embodiment, the above-mentioned peptide comprises all L-amino acids.
The peptide may also be N- and/or C-terminally capped or modified to prevent degradation, increase stability or uptake. In an embodiment, the amino terminal residue (i.e., the free amino group at the N-terminal end) of the peptide is modified (e.g., for protection against degradation), for example by covalent attachment of a moiety/chemical group (Z1). Z1 may be a straight chained or branched alkyl group of one to eight carbons, or an acyl group (R—CO—), wherein R is a hydrophobic moiety (e.g., acetyl, propionyl, butanyl, iso-propionyl, or iso-butanyl), or an aroyl group (Ar—CO—), wherein Ar is an aryl group. In an embodiment, the acyl group is a C1-C16 or C3-C16 acyl group (linear or branched, saturated or unsaturated), in a further embodiment, a saturated C1-C6 acyl group (linear or branched) or an unsaturated C3-C6 acyl group (linear or branched), for example an acetyl group (CH3—CO—, Ac). In an embodiment, Z1 is absent.
The carboxy terminal residue (i.e., the free carboxy group at the C-terminal end of the peptide) of the peptide may be modified (e.g., for protection against degradation), for example by amidation (replacement of the OH group by a NH2 group), thus in such a case Z2 is a NH2 group. In an embodiment, Z2 may be an hydroxamate group, a nitrile group, an amide (primary, secondary or tertiary) group, an aliphatic amine of one to ten carbons such as methyl amine, iso-butylamine, iso-valerylamine or cyclohexylamine, an aromatic or arylalkyl amine such as aniline, napthylamine, benzylamine, cinnamylamine, or phenylethylamine, an alcohol or CH2OH. In an embodiment, Z2 is absent.
In an embodiment, the peptide comprises the sequence ELQEKFLSL (SEQ ID NO:15), in a further embodiment the peptide is ELQEKFLSL (SEQ ID NO:15). In another embodiment, the peptide comprises the sequence ELQEKFSSL (SEQ ID NO:16), in a further embodiment the peptide is ELQEKFSSL (SEQ ID NO:16).
In an embodiment, the peptide comprises the sequence QELDGVFQKL (SEQ ID NO:17). In a further embodiment, the peptide is QELDGVFQKL (SEQ ID NO:17). In another embodiment, the peptide comprises the sequence QELDRVFQKL (SEQ ID NO:18). In a further embodiment, the peptide is QELDRVFQKL (SEQ ID NO:18).
In an embodiment, the peptide comprises the sequence SLFFRKVPF (SEQ ID NO:19). In a further embodiment, the peptide is SLFFRKVPF (SEQ ID NO:19). In another embodiment, the peptide comprises the sequence SLFFRKVAF (SEQ ID NO:20). In a further embodiment, the peptide is SLFFRKVAF (SEQ ID NO:20).
In an embodiment, the above-mentioned peptide comprises the sequence SVLKPGNSK (SEQ ID NO:21). In a further embodiment, the peptide is SVLKPGNSK (SEQ ID NO:21). In another embodiment, the above-mentioned peptide comprises the sequence TVLKPGNSK (SEQ ID NO:22). In a further embodiment, the peptide is TVLKPGNSK (SEQ ID NO:22).
In an embodiment, the above-mentioned peptide comprises the sequence AMYDKGPFRSK (SEQ ID NO:23). In an embodiment, the above-mentioned peptide is AMYDKGPFRSK (SEQ ID NO:23). In another embodiment, the above-mentioned peptide comprises the sequence AMYDKGPFVVSK (SEQ ID NO:24). In an embodiment, the above-mentioned peptide is AMYDKGPFVVSK (SEQ ID NO:24).
In an embodiment, the above-mentioned peptide comprises the sequence RVSLPTSPG (SEQ ID NO:25). In an embodiment, the above-mentioned peptide is RVSLPTSPG (SEQ ID NO:25). In another embodiment, the above-mentioned peptide comprises the sequence RVSLPTSPR (SEQ ID NO:26). In an embodiment, the above-mentioned peptide is RVSLPTSPR (SEQ ID NO:26).
In an embodiment, the above-mentioned peptide comprises the sequence VMGNPGTFK (SEQ ID NO:27). In an embodiment, the above-mentioned peptide is VMGNPGTFK (SEQ ID NO:27). In another embodiment, the above-mentioned peptide comprises the sequence VMGNPGTFN (SEQ ID NO:28). In an embodiment, the above-mentioned peptide is VMGNPGTFN (SEQ ID NO:28).
The peptides of the invention may be produced by expression in a host cell comprising a nucleic acid encoding the peptides (recombinant expression) or by chemical synthesis (e.g., solid-phase peptide synthesis). Peptides can be readily synthesized by manual and/or automated solid phase procedures well known in the art. Suitable syntheses can be performed for example by utilizing “T-boc” or “Fmoc” procedures. Techniques and procedures for solid phase synthesis are described in for example Solid Phase Peptide Synthesis: A Practical Approach, by E. Atherton and R. C. Sheppard, published by IRL, Oxford University Press, 1989. Alternatively, the peptides may be prepared by way of segment condensation, as described, for example, in Liu et al., Tetrahedron Lett. 37: 933-936, 1996; Baca et al., J. Am. Chem. Soc. 117: 1881-1887, 1995; Tam et al., Int. J. Peptide Protein Res. 45: 209-216, 1995; Schnolzer and Kent, Science 256: 221-225, 1992; Liu and Tam, J. Am. Chem. Soc. 116: 4149-4153, 1994; Liu and Tam, Proc. Natl. Acad. Sci. USA 91: 6584-6588, 1994; and Yamashiro and Li, Int. J. Peptide Protein Res. 31: 322-334, 1988). Other methods useful for synthesizing the peptides are described in Nakagawa et al., J. Am. Chem. Soc. 107: 7087-7092, 1985.
Peptides comprising naturally occurring amino acids encoded by the genetic code may also be prepared using recombinant DNA technology using standard methods.
Accordingly, in another aspect, the invention further provides a nucleic acid (isolated) encoding the above-mentioned peptides of sequences I-VII. In an embodiment, the nucleic acid does not encode the full-length CENPF, ZWINT, MTCH2, ELF1, NQO1, KIAA0226L or RMDN1 polypeptide. In an embodiment, the nucleic acid has a length of 150 nucleotides or less, in further embodiments 135, 120, 105, 90, 75, 60, 45, 42 or 39 nucleotides or less. In other embodiments, the nucleic acid comprises from about 21 nucleotides to about 45 nucleotides, from about 24 to about 36 nucleotides, for example 24, 27, 30, 33 or 36 nucleotides.
“Isolated”, as used herein, refers to a peptide or nucleic molecule separated from other components that are present in the natural source of the macromolecule (other nucleic acids, proteins, lipids, sugars, etc.). “Synthetic”, as used herein, refers to a peptide or nucleic molecule that is not isolated from its natural sources, e.g., which is produced through recombinant technology or using chemical synthesis.
In an embodiment, the above-mentioned peptide is substantially pure. A compound is “substantially pure” when it is separated from the components that naturally accompany it. Typically, a compound is substantially pure when it is at least 60%, more generally 75%, 80% or 85%, preferably over 90% and more preferably over 95%, by weight, of the total material in a sample. Thus, for example, a polypeptide that is chemically synthesized or produced by recombinant technology will generally be substantially free from its naturally associated components. A nucleic acid molecule is substantially pure when it is not immediately contiguous with (i.e., covalently linked to) the coding sequences with which it is normally contiguous in the naturally occurring genome of the organism from which the nucleic acid is derived. A substantially pure compound can be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid molecule encoding a peptide compound; or by chemical synthesis. Purity can be measured using any appropriate method such as column chromatography, gel electrophoresis, HPLC, etc.
The nucleic acid may be in a vector, such as a cloning vector or an expression vector, that may be transfected into a host cell. Alternatively, the nucleic acid may be incorporated into the genome of the host cell. In either event, the host cell expresses the nucleic acid, and in turn the encoded peptide. The invention also provides a vector or plasmid comprising the above-mentioned nucleic acid. The vector or plasmid contains the necessary elements for the transcription and translation of the inserted coding sequence, and may contain other components such as resistance genes, cloning sites, etc. Methods that are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding peptides or polypeptides and appropriate transcriptional and translational control/regulatory elements operably linked thereto. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.
“Operably linked” refers to a juxtaposition of components, particularly nucleotide sequences, such that the normal function of the components can be performed. Thus, a coding sequence that is operably linked to regulatory sequences refers to a configuration of nucleotide sequences wherein the coding sequences can be expressed under the regulatory control, that is, transcriptional and/or translational control, of the regulatory sequences. “Regulatory/control region” or “regulatory/control sequence”, as used herein, refers to the non-coding nucleotide sequences that are involved in the regulation of the expression of a coding nucleic acid. Thus the term regulatory region includes promoter sequences, regulatory protein binding sites, upstream activator sequences, and the like.
In another aspect, the present invention provides an MHC class I molecule loaded with the above-mentioned peptide. In an embodiment, the MHC molecule is a HLA-B8 molecule, in a further embodiment a HLA-B*0801 molecule. In an embodiment, the peptide is non-covalently bound to the MHC class I molecule (i.e., the peptide is loaded into the peptide binding groove/pocket but is not covalently attached to the MHC class I molecule). In another embodiment, the peptide is covalently attached/bound to the MHC class I molecule. In such a construct, the peptide and the MHC class I molecule are produced as a fusion protein, typically with a short (e.g., 5 to 20 residues, preferably about 10) flexible linker or spacer (e.g., a polyglycine linker). In another aspect, the invention provides a nucleic acid encoding a peptide-MHC class I fusion protein. In an embodiment, the MHC class I molecule-peptide complex is multimerized. Accordingly, in another aspect, the present invention provides a multimer of MHC class I molecule loaded (covalently or not) with the above-mentioned peptide. A great number of strategies have been developed for the production of MHC multimers, including MHC dimers, tetramers, pentamers, octamers, etc. (reviewed in Bakker and Schumacher, Current Opinion in Immunology 2005, 17:428-433). MHC multimers are useful, for example, for the detection and purification of antigen-specific T cells.
In yet another aspect, the present invention provides a cell (e.g., a host cell), in an embodiment an isolated cell, comprising the above-mentioned nucleic acid or vector. In another aspect, the present invention provides a cell expressing at its cell surface an MHC molecule (e.g., a HLA-B8 molecule, such as a HLA-B*0801 molecule, and/or an HLA-A3 molecule, such as a HLA-A*0301 molecule and/or an HLA-B44 allele such as HLA-B*4403) loaded with the above-mentioned peptide. In an embodiment, the host cell is a primary cell, a cell line or an immortalized cell. In another embodiment, the cell is an antigen-presenting cell (APC).
Nucleic acids and vectors can be introduced into cells via conventional transformation or transfection techniques. The terms “transformation” and “transfection” refer to techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection and viral-mediated transfection. Suitable methods for transforming or transfecting host cells can for example be found in Sambrook et al. (supra), and other laboratory manuals. Methods for introducing nucleic acids into mammalian cells in vivo are also known, and may be used to deliver the vector DNA of the invention to a subject for gene therapy.
In another embodiment, the present invention provides T cell receptor (TCR) molecules capable of interacting with the above-mentioned MHC molecule/peptide (MiHA) complex, and nucleic acid molecules encoding such TCR molecules. A TCR according to the present invention will preferably be capable of specifically interacting with an MiHA of the present invention loaded on an MHC molecule, preferably at the surface of a living cell in vitro or in vivo. T cell receptors and in particular nucleic acids encoding TCR's according to the invention may for instance be applied to transfer a TCR from one T cell to another T cell and generate new T cell clones capable of specifically recognizing the MiHA. By this TCR cloning method, T cell clones may be provided that essentially are of the genetic make-up of an allogeneic donor, for instance a donor of lymphocytes. The method to provide T cell clones capable of recognizing an MiHA according to the invention may be generated for and can be specifically targeted to tumor cells expressing the MiHA in a graft recipient, preferably an ASCT and/or donor lymphocyte infusion (DLI) recipient subject. Hence the invention provides CD8 T lymphocytes encoding and expressing a T cell receptor capable of interacting with the above-mentioned peptide/MHC molecule complex. Said T lymphocyte may be a recombinant or a naturally selected T lymphocyte. CD8 T lymphocytes of the invention may also be used for or in the methods and pharmaceutical compositions (see below). This specification thus provides at least two methods for producing CD8 T lymphocytes of the invention, comprising the step of bringing undifferentiated lymphocytes into contact with a peptide/MHC molecule complex (typically expressed at the surface of cells, such as APCs) under conditions conducive of triggering an immune response, which may be done in vitro or in vivo for instance in a patient receiving a graft. Alternatively, it may be carried out in vitro by cloning a gene encoding the TCR specific for interacting with a peptide/MHC molecule complex, which may be obtained from a cell obtained from the previous method or from a subject exhibiting an immune response against peptide/MHC molecule complex, into a host cell and/or a host lymphocyte obtained from a graft recipient or graft donor, and optionally differentiate to cytotoxic T lymphocytes (CTLs).
The potential impact of MiHA-based cancer immunotherapy is significant. For hematologic cancers (e.g., leukemia), the use of anti-MiHA T cells may replace conventional AHCT because it may provide superior anti-leukemic activity without causing GVHD. As a corollary it may benefit to many patients with hematologic malignancy who cannot be treated by conventional AHCT because their risk/reward (GVHD/GVT) ratio is too high. Finally, since studies in mice have shown that MiHA-based immunotherapy may be effective for treatment of solid tumors, MiHA-based cancer immunotherapy may be used to MiHA-targeted therapy of non-hematologic cancers, such as solid cancers.
High-avidity T cell responses capable of eradicating tumors can be generated in an allogeneic setting. In hematological malignancies, allogeneic HLA-matched hematopoietic stem cell transplantation (ASCT) provides a platform for allogeneic immunotherapy due to the induction of T cell-mediated graft-versus-tumor (GVT) immune responses. Immunotherapy in an allogeneic setting enables induction of effective T cell responses due to the fact that T cells of donor origin are not selected for low reactivity against self-antigens of the recipient. Therefore, high-affinity T cells against tumor- or recipient-specific antigens can be found in the T cell inoculum administered to the patient during or after ASCT. The main targets of the tumor-reactive T cell responses are polymorphic proteins for which donor and recipient are disparate, namely MiHAs. The MiHA peptide sequences identified herein may be used for the production of synthetic peptides to be used i) for in vitro priming and expansion of MiHA-specific T cells to be injected into transplant (AHCT) recipients and/or ii) as vaccines to boost the graft-vs.-tumor effect (GVTE) in recipients of MiHA-specific T cells, subsequent to the transplantation.
In another aspect, the present invention provides the use of the above-mentioned peptide of sequences (I) to (VII) in the immunotherapy of cancer.
Accordingly, in another aspect, the present invention provides a method of treating cancer, said method comprising administering to a subject in need thereof an effective amount of CD8 T lymphocytes recognizing a MHC class I molecule loaded with the above-mentioned peptide.
In another aspect, the present invention provides the use of CD8 T lymphocytes recognizing a MHC class I molecule loaded with the above-mentioned peptide for treating cancer in a subject. In another aspect, the present invention provides the use of CD8 T lymphocytes recognizing a MHC class I molecule loaded with the above-mentioned peptide for the preparation/manufacture of a medicament for treating cancer in a subject. In an embodiment, the subject is a transplant (e.g., AHCT) recipient.
In an embodiment, the above-mentioned method or use further comprises determining whether said subject expresses a CENPF nucleic acid comprising a T or a C at a position corresponding to nucleotide 4409 in the nucleic acid sequence of human CENPF (FIGS. 1A to 1C, NCBI Reference Sequence: NM_016343.3), and/or a CENPF polypeptide comprising a leucine or serine residue at a position corresponding to residue 1412 in the protein sequence of human CENPF (FIG. 1D, NCBI Reference Sequence: NP_057427.3), wherein (a) if said subject expresses a CENPF nucleic acid comprising a T at a position corresponding to nucleotide 4409 in the nucleic acid sequence of human CENPF and/or a CENPF polypeptide comprising a leucine residue at a position corresponding to residue 1412 in the protein sequence of human CENPF, X2 is L in the peptide; (b) if said subject expresses a CENPF nucleic acid comprising a C at a position corresponding to nucleotide 4409 in the nucleic acid sequence of human CENPF and/or a CENPF polypeptide comprising a serine residue at a position corresponding to residue 1412 in the protein sequence of human CENPF, X2 is S in the peptide.
In an embodiment, the above-mentioned method or use further comprises determining whether said subject expresses a ZWINT nucleic acid comprising an A or a G at a position corresponding to nucleotide 596 in the nucleic acid sequence of human ZWINT (FIG. 2A, NCBI Reference Sequence: NM_007057.3), and/or a ZWINT polypeptide comprising a arginine or glycine residue at a position corresponding to residue 187 in the protein sequence of human ZWINT (FIG. 2B, NCBI Reference Sequence: NP_008988.2), wherein (a) if said subject expresses a ZWINT nucleic acid comprising an A at a position corresponding to nucleotide 596 in the nucleic acid sequence of human ZWINT and/or a ZWINT polypeptide comprising an arginine residue at a position corresponding to residue 187 in the protein sequence of human ZWINT, X5 is R in said peptide; (b) if said subject expresses a ZWINT nucleic acid comprising a G at a position corresponding to nucleotide 596 in the nucleic acid sequence of human ZWINT and/or a ZWINT polypeptide comprising a glycine residue at a position corresponding to residue 187 in the protein sequence of human ZWINT, X5 is G in said peptide.
In an embodiment, the above-mentioned method further comprises determining whether said subject expresses a MTCH2 nucleic acid comprising a C or a G at a position corresponding to nucleotide 1057 in the nucleic acid sequence of human MTCH2 (FIG. 3A, NCBI Reference Sequence: NM_014342.3), and/or a MTCH2 polypeptide comprising a proline or alanine residue at a position corresponding to residue 290 in the protein sequence of human MTCH2 (FIG. 3B, NCBI Reference Sequence: NP_055157.1), wherein (a) if said subject expresses a MTCH2 nucleic acid comprising a C at a position corresponding to nucleotide 1057 in the nucleic acid sequence of human MTCH2 and/or a MTCH2 polypeptide comprising a proline residue at a position corresponding to residue 290 in the protein sequence of human MTCH2, X9 is P in said peptide; (b) if said subject expresses a MTCH2 nucleic acid comprising a G at a position corresponding to nucleotide 1057 in the nucleic acid sequence of human MTCH2 and/or a MTCH2 polypeptide comprising an alanine residue at a position corresponding to residue 290 in the protein sequence of human MTCH2, X5 is A in said peptide.
In an embodiment, the above-mentioned method further comprises determining whether said subject expresses an ELF1 nucleic acid comprising an A or T at a position corresponding to nucleotide 1400 in the nucleic acid sequence of human ELF1 (FIGS. 4A and 4B, NCBI Reference Sequence: NM_172373.3) and/or an ELF1 polypeptide having a threonine or a serine at a position corresponding to residue 343 in the ELF1 protein sequence (FIG. 4C, NCBI Reference Sequence: NP_758961.1), wherein (a) if said subject expresses an ELF1 nucleic acid comprising an A at a position corresponding to nucleotide 1400 in the nucleic acid sequence of human ELF1 and/or an ELF1 polypeptide comprising a threonine residue at a position corresponding to residue 343 in the protein sequence of human ELF1, X12 is T in said peptide; (b) if said subject expresses an ELF1 nucleic acid comprising a T at a position corresponding to nucleotide 1400 in the nucleic acid sequence of human ELF1 and/or an ELF1 polypeptide comprising a serine residue at a position corresponding to residue 343 in the protein sequence of human ELF1, X12 is S in said peptide.
In an embodiment, the above-mentioned method further comprises determining whether said subject expresses an NQO1 nucleic acid comprising an C or T at a position corresponding to nucleotide 615 in the nucleic acid sequence of human NQO1 (FIG. 5A, NCBI Reference Sequence: NM_000903.2) and/or an NQO1 polypeptide having an arginine or a tryptophan at a position corresponding to residue 139 in the NQO1 protein sequence (FIG. 5B, NCBI Reference Sequence: NP_000894.1), wherein (a) if said subject expresses a NQO1 nucleic acid comprising an C at a position corresponding to nucleotide 615 in the nucleic acid sequence of human NQO1 and/or a NQO1 polypeptide comprising an arginine residue at a position corresponding to residue 139 in the protein sequence of human NQO1, X17 is R in said peptide; (b) if said subject expresses an NQO1 nucleic acid comprising a T at a position corresponding to nucleotide 615 in the nucleic acid sequence of human NQO1 and/or a NQO1 polypeptide comprising a tryptophan residue at a position corresponding to residue 139 in the protein sequence of human NQO1, X17 is W in said peptide.
In an embodiment, the above-mentioned method further comprises determining whether said subject expresses a KIAA0226L nucleic acid comprising a G or A at a position corresponding to nucleotide 1059 in the nucleic acid sequence of human KIAA0226L (FIGS. 6A and 6B, NCBI Reference Sequence: NM_025113.2) and/or an KIAA0226L polypeptide having a glycine or an arginine at a position corresponding to residue 152 in the KIAA0226L protein sequence (FIG. 6C, NCBI Reference Sequence: NP_079389.2), wherein (a) if said subject expresses a KIAA0226L nucleic acid comprising a G at a position corresponding to nucleotide 1059 in the nucleic acid sequence of human KIAA0226L and/or a KIAA0226L polypeptide comprising a glycine residue at a position corresponding to residue 152 in the protein sequence of human KIAA0226L, X23 is G in said peptide; (b) if said subject expresses a KIAA0226L nucleic acid comprising an A at a position corresponding to nucleotide 1059 in the nucleic acid sequence of human a KIAA0226L and/or a KIAA0226L polypeptide comprising an arginine residue at a position corresponding to residue 152 in the protein sequence of human KIAA0226L, X23 is R in said peptide.
In an embodiment, the above-mentioned method further comprises determining whether said subject expresses an RMDN1 nucleic acid comprising an A or C at a position corresponding to nucleotide 316 in the nucleic acid sequence of human RMDN1 (FIG. 7A, NCBI Reference Sequence: NM_016033.2) and/or an RMDN1 polypeptide having a lysine or an asparagine at a position corresponding to residue 52 in the RMDN1 protein sequence (FIG. 7B, NCBI Reference Sequence: NP_057117.2), wherein (a) if said subject expresses an RMDN1 nucleic acid comprising an A at a position corresponding to nucleotide 316 in the nucleic acid sequence of human RMDN1 and/or an RMDN1 polypeptide comprising a lysine residue at a position corresponding to residue 52 in the protein sequence of human RMDN1, X28 is K in said peptide; (b) if said subject expresses an RMDN1 nucleic acid comprising a C at a position corresponding to nucleotide 316 in the nucleic acid sequence of human RMDN1 and/or a RMDN1 polypeptide comprising an asparagine residue at a position corresponding to residue 52 in the protein sequence of human RMDN1, X28 is N in said peptide.
The above-noted polymorphism (nucleotide variation) in the nucleic acid and/or protein of interest (e.g., CENPF, ZWINT, MTCH2, ELF1, NQO1, KIAA0226L or RMDN1) may be detected by a number of methods which are known in the art. Examples of suitable methods for detecting alterations at the nucleic acid level include sequencing of the nucleic acid sequence of the nucleic acid of interest (e.g., CENPF, ZWINT, MTCH2, ELF1, NQO1, KIAA0226L or RMDN1); hybridization of a nucleic acid probe capable of specifically hybridizing to a nucleic acid of interest (e.g., CENPF, ZWINT, MTCH2, ELF1, NQO1, KIAA0226L or RMDN1) comprising the polymorphism (the first allele) and not to (or to a lesser extent to) a corresponding nucleic acid that do not comprise the polymorphism (the second allele) (under comparable hybridization conditions, such as stringent hybridization conditions), or vice-versa; restriction fragment length polymorphism analysis (RFLP); Amplified fragment length polymorphism PCR (AFLP-PCR); amplification of a nucleic acid fragment using a primer specific for one of the allele, wherein the primer produces an amplified product if the allele is present and does not produce the same amplified product when the other allele is used as a template for amplification (e.g., allele-specific PCR). Other methods include in situ hybridization analyses and single-stranded conformational polymorphism analyses. Further, nucleic acids of interest (e.g., CENPF, ZWINT, MTCH2, ELF1, NQO1, KIAA0226L or RMDN1 nucleic acids) may be amplified using known methods (e.g., polymerase chain reaction [PCR]) prior to or in conjunction with the detection methods noted herein. The design of various primers for such amplification is known in the ad. The nucleic acid (mRNA) may also be reverse transcribed into cDNA prior to analysis.
Examples of suitable methods for detecting alterations/polymorphisms at the polypeptide level include sequencing of the polypeptide of interest (e.g., CENPF, ZWINT, MTCH2, ELF1, NQO1, KIAA0226L or RMDN1); digestion of the polypeptide followed by mass spectrometry or HPLC analysis of the peptide fragments, wherein the alteration/polymorphism of the polypeptide of interest (e.g., CENPF, ZWINT, MTCH2, ELF1, NQO1, KIAA0226L or RMDN1) results in an altered mass spectrometry or HPLC spectrum as compared to the native polypeptide of interest (e.g., CENPF, ZWINT, MTCH2, ELF1, NQO1, KIAA0226L or RMDN1); and immunodetection using an immunological reagent (e.g., an antibody, a ligand) which exhibits altered immunoreactivity with a polypeptide comprising the alteration (first allele) relative to a corresponding native polypeptide not comprising the alteration (second allele), for example by targeting an epitope comprising the amino acid change. Immunodetection can measure the amount of binding between a polypeptide molecule and an anti-protein antibody by the use of enzymatic, chromodynamic, radioactive, magnetic, or luminescent labels which are attached to either the anti-protein antibody or a secondary antibody which binds the anti-protein antibody. In addition, other high affinity ligands may be used. Immunoassays which can be used include e.g. ELISAs, Western blots, and other techniques known to those of ordinary skill in the art (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999 and Edwards R, Immunodiagnostics: A Practical Approach, Oxford University Press, Oxford; England, 1999).
All these detection techniques may also be employed in the format of microarrays, protein-arrays, antibody microarrays, tissue microarrays, electronic biochip or protein-chip based technologies (see Schena M., Microarray Biochip Technology, Eaton Publishing, Natick, Mass., 2000).
Further, nucleic acids of interest (e.g., CENPF, ZWINT, MTCH2, ELF1, NQO1, KIAA0226L or RMDN1 nucleic acids) may be amplified using known methods (e.g., polymerase chain reaction [PCR]) prior to or in conjunction with the detection methods noted herein. The design of various primers for such amplification is known in the art.
In an embodiment, the above determining comprises sequencing in a biological sample from a subject a region of a nucleic acid corresponding to the region encompassing (i) nucleotide 4409 of a human CENPF nucleic acid (SEQ ID NO:1), (ii) nucleotide 596 in the nucleic acid of a human ZWINT nucleic acid (SEQ ID NO:3), (iii) nucleotide 1057 in the nucleic acid of a human MTCH2 nucleic acid (SEQ ID NO:5), (iv) nucleotide 1400 in the nucleic acid of a human ELF1 nucleic acid (SEQ ID NO:7), (v) nucleotide 615 in the nucleic acid of a human NQO1 nucleic acid (SEQ ID NO:9), (vi) nucleotide 1059 in the nucleic acid of a human KIAA0226L nucleic acid (SEQ ID NO:11) and/or (vii) nucleotide 316 in the nucleic acid of a human RMDN1 nucleic acid (SEQ ID NO:13).
In an embodiment, the above-mentioned CD8 T lymphocytes are in vitro expanded CD8 T lymphocytes. Expanded CD8 T lymphocytes may be obtained by culturing primary CD8 T lymphocytes (from a donor) under conditions permitting the proliferation and/or differentiation of the CD8 T lymphocytes. Such conditions typically include contacting the CD8 T lymphocytes with cells, such as APCs, expressing at their surface peptide/MHC complexes, in the presence of growth factors and/or cytokines such as IL-2, IL-7 and/or IL-15 (see, e.g., Montes et al., Clin Exp Immunol. 2005 November; 142(2):292-302). Such expanded CD8 T lymphocytes are then administered to the recipient, for example through intravenous infusion.
In an embodiment, the subject is an allogeneic stem cell transplantation (ASCT) or donor lymphocyte infusion (DLI) recipient.
In another aspect, the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising (a) determining whether a candidate donor expresses a CENPF nucleic acid comprising a T or a C at a position corresponding to nucleotide 4409 in the nucleic acid sequence of human CENPF (FIGS. 1A to 1D, NCBI Reference Sequence: NM_016343.3), and/or a CENPF polypeptide comprising a leucine or serine residue at a position corresponding to residue 1412 in the protein sequence of human CENPF (FIG. 1E, NCBI Reference Sequence: NP_057427.3) and (b)(i) if said candidate donor expresses a CENPF nucleic acid comprising a T at a position corresponding to nucleotide 4409 in the nucleic acid sequence of human CENPF and/or a CENPF polypeptide comprising a leucine residue at a position corresponding to residue 1412 in the protein sequence of human CENPF, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-B*0801 allele loaded with the above-mentioned peptide, wherein X2 is S in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (b)(ii) if said candidate donor expresses a CENPF nucleic acid comprising a C at a position corresponding to nucleotide 4409 in the nucleic acid sequence of human CENPF and/or a CENPF polypeptide comprising a serine residue at a position corresponding to residue 1412 in the protein sequence of human CENPF, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-B*0801 allele loaded with the above-mentioned peptide, wherein X2 is L in said peptide, under conditions suitable for CD8 T lymphocyte expansion.
In another aspect, the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising: (a) determining whether a candidate donor expresses a ZWINT nucleic acid comprising an A or a G at a position corresponding to nucleotide 596 in the nucleic acid sequence of human ZWINT (FIG. 2A, NCBI Reference Sequence: NM_007057.3), and/or a ZWINT polypeptide comprising a arginine or glycine residue at a position corresponding to residue 187 in the protein sequence of human ZWINT (FIG. 2B, NCBI Reference Sequence: NP_008988.2) and (b)(i) if said candidate donor expresses a ZWINT nucleic acid comprising an A at a position corresponding to nucleotide 596 in the nucleic acid sequence of human ZWINT and/or a ZWINT polypeptide comprising an arginine residue at a position corresponding to residue 187 in the protein sequence of human ZWINT, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-B*4403 allele loaded with the above-mentioned peptide (II), wherein X5 is G in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (b)(ii) if said candidate donor expresses a ZWINT nucleic acid comprising a G at a position corresponding to nucleotide 596 in the nucleic acid sequence of human ZWINT and/or a ZWINT polypeptide comprising a glycine residue at a position corresponding to residue 187 in the protein sequence of human ZWINT, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-B*4403 allele loaded with the above-mentioned peptide (II), wherein X5 is R in said peptide, under conditions suitable for CD8 T lymphocyte expansion.
In another aspect, the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising: (a) determining whether a candidate donor expresses a MTCH2 nucleic acid comprising a C or a G at a position corresponding to nucleotide 1057 in the nucleic acid sequence of human MTCH2 (FIG. 3A, NCBI Reference Sequence: NM_014342.3), and/or a MTCH2 polypeptide comprising a proline or alanine residue at a position corresponding to residue 290 in the protein sequence of human MTCH2 (FIG. 3B, NCBI Reference Sequence: NP_055157.1) and (b)(i) if said candidate donor expresses a MTCH2 nucleic acid comprising a C at a position corresponding to nucleotide 1057 in the nucleic acid sequence of human MTCH2 and/or a MTCH2 polypeptide comprising a proline residue at a position corresponding to residue 290 in the protein sequence of human MTCH2, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-B0801 allele loaded with the peptide, wherein X9 is A in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (b)(ii) if said candidate donor expresses a MTCH2 nucleic acid comprising a G at a position corresponding to nucleotide 1057 in the nucleic acid sequence of human MTCH2 and/or a MTCH2 polypeptide comprising an alanine residue at a position corresponding to residue 290 in the protein sequence of human MTCH2, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-B*0801 allele loaded with the peptide, wherein X9 is P in said peptide, under conditions suitable for CD8 T lymphocyte expansion.
In another aspect, the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising: (a) determining whether a candidate donor expresses an ELF1 nucleic acid comprising an A or T at a position corresponding to nucleotide 1400 in the nucleic acid sequence of human ELF1 (FIGS. 4A and 4B, NCBI Reference Sequence: NM_172373.3) and/or an ELF1 polypeptide having a threonine or a serine at a position corresponding to residue 343 in the ELF1 protein sequence (FIG. 4C, NCBI Reference Sequence: NP_758961.1) and (b)(i) if said candidate donor expresses an ELF1 nucleic acid comprising an A at a position corresponding to nucleotide 1400 in the nucleic acid sequence of human ELF1 and/or an ELF1 polypeptide having a threonine at a position corresponding to residue 343 in the ELF1 protein sequence, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the above-mentioned peptide (IV), wherein X12 is S in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (b)(ii) if said candidate donor expresses an ELF1 nucleic acid comprising a T at a position corresponding to nucleotide 1400 in the nucleic acid sequence of human ELF1 and/or an ELF1 polypeptide having a serine at a position corresponding to residue 343 in the ELF1 protein sequence, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the above-mentioned peptide (IV), wherein X12 is T in said peptide, under conditions suitable for CD8 T lymphocyte expansion.
In another aspect, the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising: (a) determining whether said subject expresses an NQO1 nucleic acid comprising a C or T at a position corresponding to nucleotide 615 in the nucleic acid sequence of human NQO1 (FIG. 5A, NCBI Reference Sequence: NM_000903.2) and/or an NQO1 polypeptide having an arginine or a tryptophan at a position corresponding to residue 139 in the NQO1 protein sequence (FIG. 5B, NCBI Reference Sequence: NP_000894.1) and (b)(i) if said candidate donor expresses a NQO1 nucleic acid comprising a C at a position corresponding to nucleotide 615 in the nucleic acid sequence of human NQO1 and/or a NQO1 polypeptide comprising an arginine residue at a position corresponding to residue 139 in the protein sequence of human NQO1, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the above-mentioned peptide (V), wherein X17 is W in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (b)(ii) if said candidate donor expresses a NQO1 nucleic acid comprising a T at a position corresponding to nucleotide 615 in the nucleic acid sequence of human NQO1 and/or a NQO1 polypeptide comprising a tryptophan residue at a position corresponding to residue 139 in the protein sequence of human NQO1, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the above-mentioned peptide (V), wherein X17 is R in said peptide, under conditions suitable for CD8 T lymphocyte expansion.
In another aspect, the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising: (a) determining whether said subject expresses a KIAA0226L nucleic acid comprising a G or A at a position corresponding to nucleotide 1059 in the nucleic acid sequence of human KIAA0226L (FIGS. 6A and 6B, NCBI Reference Sequence: NM_025113.2) and/or a KIAA0226L polypeptide having a glycine or an arginine at a position corresponding to residue 152 in the KIAA0226L protein sequence (FIG. 6C, NCBI Reference Sequence: NP_079389.2) and (b)(i) if said candidate donor expresses a KIAA0226L nucleic acid comprising a G at a position corresponding to nucleotide 1059 in the nucleic acid sequence of human KIAA0226L and/or a KIAA0226L polypeptide comprising a glycine residue at a position corresponding to residue 152 in the protein sequence of human KIAA0226L, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the above-mentioned peptide (VI), wherein X23 is R in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (b)(ii) if said candidate donor expresses a KIAA0226L nucleic acid comprising an A at a position corresponding to nucleotide 1059 in the nucleic acid sequence of human KIAA0226L and/or a KIAA0226L polypeptide comprising an arginine residue at a position corresponding to residue 152 in the protein sequence of human KIAA0226L, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the above-mentioned peptide (VI), wherein X23 is G in said peptide, under conditions suitable for CD8 T lymphocyte expansion.
In another aspect, the present invention provides a method of expanding CD8 T lymphocytes for adoptive T-cell immunotherapy, said method comprising: (a) determining whether said subject expresses an RMDN1 nucleic acid comprising an A or C at a position corresponding to nucleotide 316 in the nucleic acid sequence of human RMDN1 (FIG. 7A, NCBI Reference Sequence: NM_016033.2) and/or an RMDN1 polypeptide having a lysine or an asparagine at a position corresponding to residue 52 in the RMDN1 protein sequence (FIG. 7B, NCBI Reference Sequence: NP_057117.2) and (b)(i) if said candidate donor expresses a RMDN1 nucleic acid comprising a A at a position corresponding to nucleotide 316 in the nucleic acid sequence of human RMDN1 and/or a RMDN1 polypeptide comprising a lysine residue at a position corresponding to residue 52 in the protein sequence of human RMDN1, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the above-mentioned peptide (VII), wherein X28 is N in said peptide, under conditions suitable for CD8 T lymphocyte expansion; or (b)(ii) if said candidate donor expresses a RMDN1 nucleic acid comprising a C at a position corresponding to nucleotide 316 in the nucleic acid sequence of human RMDN1 and/or a RMDN1 polypeptide comprising an arginine residue at a position corresponding to residue 52 in the protein sequence of human RMDN1, culturing CD8 T lymphocytes from said candidate donor in the presence of cells expressing a MHC class I molecule of the HLA-A*0301 allele loaded with the above-mentioned peptide (VII), wherein X28 is K in said peptide, under conditions suitable for CD8 T lymphocyte expansion.
In an embodiment, the above-mentioned cancer comprises tumor cells expressing CENPF, ZWINT, MTCH2, ELF1, NQO1, KIAA0226L and/or RMDN1.
In an embodiment, the above-mentioned cancer comprises tumor cells expressing CENPF, and is a hematopoietic cancer, such as leukemia, lymphoma, or myeloma, or a solid tumor such as head and neck squamous cell carcinomas, breast cancer, non-Hodgkin's lymphoma or gastrointestinal cancer.
In another embodiment, the above-mentioned cancer comprises tumor cells expressing ZWINT, and is a breast, prostate or bladder cancer.
In another embodiment, the above-mentioned cancer comprises tumor cells expressing MTCH2, and is a solid tumor/cancer, in a further embodiment lung, thyroid, liver, esophagus, colon or breast cancer, or osteosarcoma.
In another embodiment, the above-mentioned cancer comprises tumor cells expressing ELF1, and is leukemia, lung cancer (e.g., non-small cell lung cancer), breast cancer or ovarian cancer.
In another embodiment, the above-mentioned cancer comprises tumor cells expressing NQO1, and is lung cancer (e.g., non-small cell lung cancer), skin cancer, breast cancer, liver cancer (e.g., intrahepatic cholangiocarcinoma) digestive tract cancer such as colorectal cancer, or pancreatic cancer.
In another embodiment, the above-mentioned cancer comprises tumor cells expressing KIAA0226L, and is a lymphoma (e.g., Burkitt's lymphoma), skin cancer, breast cancer, liver cancer (e.g., intrahepatic cholangiocarcinoma) digestive tract cancer such as colorectal cancer, or pancreatic cancer.
Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims. In the claims, the word “comprising” is used as an open-ended term, substantially equivalent to the phrase “including, but not limited to”. The singular forms “a”, “an” and the include corresponding plural references unless the context clearly dictates otherwise.
MODE(S) FOR CARRYING OUT THE INVENTION
The present invention is illustrated in further details by the following non-limiting examples.
Example 1: Materials and Methods
Cell Culture.
Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples of 2 HLA-identical siblings. Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (B-LCLs) were derived from PBMCs with Ficoll-Paque™ Plus (Amersham) as described (Tosato and Cohen, 2007). Established B-LCLs were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 25 mM of HEPES, 2 mM L-glutamine and penicillin-streptomycin (all from Invitrogen).
HLA Typing.
High-resolution HLA genotyping was performed at the Maisonneuve-Rosemont Hospital. The HLA genotype of our subjects was HLA-A*0301/2902, HLA-B*0801/*4403, HLA-C*0701/*1601 and HLA-DRB1*0301/*0701.
RNA Extraction.
Total RNA was isolated from 5 million B-LCLs using RNeasy™ mini kit including DNase I treatment (Qiagen) according to the manufacturer's instructions. Total RNA was quantified using the NanoDrop™ 2000 (Thermo Scientific) and RNA quality was assessed with the 2100 Bioanalyzer™ (Agilent Technologies).
Preparation of Transcriptome Libraries.
Transcriptome libraries were generated from 1 μg of total RNA using the TruSeq™ RNA Sample Prep Kit (v2) (RS-930-1021, Illumina) following the manufacturer's protocol. Briefly, poly-A mRNA was purified using poly-T oligo-attached magnetic beads using two rounds of purification. During the second elution of the poly-A RNA, the RNA was fragmented and primed for cDNA synthesis. Reverse transcription (RT) of the first strand was done using random primers and SuperScript™ II (InvitroGene). A second round of RT was also done to generate a double-stranded cDNA, which was then purified using Agencourt AMpure™ XP PCR purification system (Beckman Coulter). End repair of fragmented cDNA, adenylation of the 3′ ends and ligation of adaptors were done following the manufacturer's protocol. Enrichment of DNA fragments containing adapter molecules on both ends was done using 15 cycles of PCR amplification and the Illumina™ PCR mix and primers cocktail.
DNA Extraction.
Genomic DNA was extracted from 5 million B-LCLs using the PureLink™ Genomic DNA Mini Kit (Invitrogen) according to the manufacturer's instructions. DNA was quantified and quality-assessed using the NanoDrop™ 2000 (Thermo Scientific).
Preparation of Genomic DNA Libraries and Exome Enrichment.
Genomic libraries were constructed from 1 μg of genomic DNA using the TruSeq™ DNA Sample Preparation Kit (v2) (FC-930-1021, Illumina) following the manufacturer's protocol. This included the following steps: DNA shearing using a Covaris™ S2 instrument, DNA end repair, 3′ ends adenylation, ligation of adaptors, purification of ligation products and PCR amplification to enrich DNA fragments that have adapter molecules.
DNA-Seq libraries were subjected to an enrichment step to sequence only the coding portion (exome) of the human genome. 500 ng of DNA-Seq libraries were used for hybrid selection-based exome enrichment with the TruSeq™ exome enrichment kit (FC-930-1012, Illumina) according to the manufacturer's instructions.
Whole Transcriptome Sequencing (RNA-Seq) and Exome Sequencing.
Paired-end (2×100 bp) sequencing was performed using the Illumina HiSeq2000™ machine running TruSeq™ v3 chemistry. Cluster density was targeted at around 600-800 k clusters/mm2. Two RNA-Seq or four exomes libraries were sequenced per lane (8 lanes per slide). Detail of the Illumina sequencing technologies can be found at http://www.illumina.com/applications/detail/sequencinq/dna_sequencing.ilmn. Briefly, DNA or RNA libraries are incorporated into a fluidic flow cell design with 8 individual lanes. The flow cell surface is populated with capture oligonucleotide anchors, which hybridize the appropriately modified DNA segments of a sequencing library. By a process called “bridge amplification,” captured DNA templates are amplified in the flow cell by “arching” over and hybridizing to an adjacent anchor oligonucleotide primer. The sequencing reaction is performed by hybridizing a primer complementary to the adapter sequence and then cyclically adding DNA polymerase and a mixture of 4 differently colored fluorescent reversible dye terminators to the captured DNA in the flow cell. By using this approach, unmodified DNA fragments and unincorporated nucleotides are washed away, while captured DNA fragments are extended one nucleotide at a time. After each nucleotide-coupling cycle takes place, the flow cell is scanned, and digital images are acquired to record the locations of fluorescently labeled nucleotide incorporations. Following imaging, the fluorescent dye and the terminal 3 blocker are chemically removed from the DNA before the next nucleotide coupling cycle.
Read Mapping.
Sequence data were mapped to the human reference genome (hg19) using the Ilumina Casava™ 1.8.1 and the Eland™ v2 mapping softwares. First, the *.bcl files were converted into compressed FASTQ files, following by demultiplexing of separate multiplexed sequence runs by index. Then, single reads were aligned to the human reference genome using the multiseed and gapped alignment method. Multiseed alignment works by aligning the first seed of 32 bases and consecutive seeds separately. Gapped alignment extends each candidate alignment to the full length of the read and allows for gaps up to 10 bases. The following criteria were applied: i) a read has to have at least one seed that matches with at most 2 mismatches, and for that seed no gaps are allowed and ii) for the whole read any number of gaps were allowed, as long as they correct at least five mismatches downstream. For each candidate alignment a probability score was calculated. This score is based on the sequencing base quality values and the positions of the mismatches. The alignment score of a read, which is expressed on the Phred scale, was computed from the probability scores of the candidate alignments. The best alignment for a given read corresponded to the candidate alignment with the highest probability score and it was picked if the alignment score exceeded a threshold. Reads that mapped at 2 or more locations (multireads) were not included in further analyses. An additional alignment was done against splice junctions and contaminants (mitochondrial and ribosomal RNA).
Identification of Single Nucleotide Variations.
The first step of the process consists of retrieving the list of all single nucleotide variations (SNV) observed between the reference genome (GRCh37.p2, NCBI) and the sequenced transcriptome of each of our subjects. This was done by using the SNP calling program Casava™ v1.8.2 from Ilumina (http://support.illumina.com/sequencing/sequencing_software/casava.ilmn). Casava calculates and retrieves statistics about every observed SNV including its position, the reference base, the raw counts for each base, the most probable genotype (max_gt) and the probability of the most probable genotype (Qmax_gt). Among the SNVs called by Casava™ only those that had a high confidence (Qmax_gt value>20) were considered. SNVs with Qmax_gt value below this threshold were assigned with the reference base instead. This strategy was used to identify SNVs at the transcript level between each of our subjects and the reference genome.
In Silico Translated Transcriptome.
The sequences containing the identified SNVs of each individual were further processed. For each sequence, all transcripts reported in Ensembl (http://useast.ensembl.org/info/data/ftp/index.html Flicek et al., Ensembl 2012, Nucleic Acids Research 2012 40 Database issue: D84-D90) were retrieved and in silico translated each of them into proteins using our in-house software pyGeno (Granados et al., 2012). The in silico translated transcriptomes included cases in which more than one non-synonymous allele was found for a given position. Supposing that a polymorphic position could affect 11mers or smaller peptides upstream or downstream, a window of 66 bp around each one of these polymorphic positions was considered and computed every possible amino acid (aa) sequence variant defined by these 66 bp (or 22 aa) windows. In this way, a list of most possible aa sequences of at most 11 aa affected by non-synonymous polymorphisms was obtained. The number of aa sequences affected by one non-synonymous polymorphism was limited to 10240 to limit the size of the file. All translated sequences were compiled into a single FASTA file that was used as a database for the identification of MHC class I-associated peptides (see “MS/MS sequencing and peptide clustering” section).
Mass Spectrometry and Peptide Sequencing.
Three biological replicates of 4×108 exponentially growing B-LCLs were prepared from each subject. MHC class I-associated peptides were released by mild acid treatment, pretreated by desalting with an HLB cartridge, filtered with a 3000 Da cut-off column and separated by cation exchange chromatography (SCX) using an off-line 1100 series binary LC system (Agilent Technologies) as previously described (Fortier et al., 2008). Peptides were loaded at 8 uL/min on a homemade strong cation exchange (SCX) column (0.3 mm internal diameter×50 mm length) packed with SCX bulk material (Polysulfoethyl A™, PolyLC). Peptides were separated into five fractions using a linear gradient of 0-25% B in 25 min (solvent A: 5 mM ammonium formate, 15% acetonitrile, ACN, pH 3.0; solvent B: 2 M ammonium formate, 15% ACN, pH 3.0) and brought to dryness using a Speedvac.
MHC class I-associated peptide fractions were resuspended in 2% aqueous ACN (0.2% formic acid) and analyzed by LC-MS/MS using an Eksigent™ LC system coupled to a LTQ-Orbitrap™ mass spectrometer (Thermo Electron) (Fortier et al., 2008; de Verteuil et al., 2010; Caron et al., 2011). Peptides were separated in a custom C18 reversed phase column (150 μm i.d.×100 mm, Jupiter Proteo™ 4 μm, Phenomenex) at a flow rate of 600 nL/min using a linear gradient of 3-60% aqueous ACN (0.2% formic acid) in 69 mins. Full mass spectra were acquired with the Orbitrap™ analyzer operated at a resolving power of 60 000 (at m/z 400) and collision-activated dissociation tandem mass spectra were acquired in data-dependent mode with the linear ion trap analyzer. Mass calibration used an internal lock mass (protonated (Si(CH3)2O))6; m/z 445.120029) and mass accuracy of peptide measurements was within 5 ppm.
MS/MS Sequencing and Peptide Clustering.
Mass spectra were analyzed using Xcalibur™ software and peak lists were generated using Mascot™ distiller version 2.1.1 (Matrix Science, http://www.matrixscience.com). Database searches were performed against a non-redundant human Uniprot database (containing 110,361 sequences, released on 28 Jul. 2011) (version 101 of the UniProt Gene Ontology Annotation available from the European Bioinformatics Institute (EBI), an academic research institute located on the Wellcome Trust Genome Campus in Hinxton near Cambridge (UK), part of the European Molecular Biology Laboratory; http://www.ebi.ac.uk/Information/. Magrane et al., Database Vol. 2011, Article ID bar009; The UniProt Consortium, Nucleic Acids Res. 2011 January; 39(Database issue): D214-D219) and databases specific for each individual (see “in silico translated transcriptome” section) using Mascot (version 2.3, Matrix Science). A Mascot search against a concatenated target/decoy database consisting of combined forward and reverse versions of the Ensembl human reference genome database and of each subject-specific database. Non-redundant peptide sequences with a cutoff score threshold above 15 were selected. The tolerances for precursor and fragment mass values were set to 0.02 and 0.05 Dalton, respectively. Searches were performed without enzyme specificity and a variable modification for oxidation (Met) and deamidation (Asn, Gln). Raw data files were converted to peptide maps comprising m/z values, charge state, retention time and intensity for all detected ions above a threshold of 8000 counts using in-house software (Proteoprofile) (Fortier et al., 2008; de Verteuil et al., 2010; Caron et al., 2011). Peptide maps were aligned together and peptide ions of each map (including non-sequenced ions) were aggregated to their corresponding Mascot identification, creating a peptide abundance profile. When multiple Mascot identifications were associated to the same ions, only the one with the highest score was kept. The intensity counts were then summed for identical peptide sequences resulting in a non-redundant abundance profile of identified peptide sequences.
Selection of MiHA Candidates and Identification of Proteins Source of MiHAs.
Peptides were filtered by their length and those peptides with the canonical MHC I-associated peptide length (typically 8-11 mers) were kept. Peptides were considered to be undetected/not expressed if they were absent in 3 replicates per subject, and detected/expressed if they were identified in at least 2 replicates per subject. The predicted binding affinity (IC50) of peptides to the allelic products was obtained using NetMHCcons version 1.0 (http://www.cbs.dtu.dk/services/NetMHCcons/) (Karosiene et al., 2011) and was used to classify MHC I peptides. Peptides with an IC50 below 50 nM were considered as strong binders and peptides with an IC50 between 50 and 500 nM were considered as weak binders.
MiHA peptides were selected according to the following criteria:
    • i) Presence of a non-synonymous SNV between the 2 subjects in the peptide-coding region leading to the exclusive surface expression of the corresponding peptide(s) in one of the 2 subjects. These constitute MiHA differences between the subjects.
    • ii) Presence of a reported non-synonymous SNP in the peptide-coding region of the subjects leading to surface expression of the corresponding peptide(s). These constitute MiHA differences between the subjects and other individuals harboring the alternate allele for the reported SNP.
The RNA (cDNA) and DNA sequences encoding MiHAs candidates were manually inspected using the Integrative Genomics Viewer v2.0 (The Broad Institute). The UCSC Repeat Masker track was included to discard candidates that corresponded to repetitive regions. A dbSNP (build 135) track was also used to identify those SNVs in the MiHA-encoding region that corresponded to reported SNPs. MiHA candidates were further inspected for mass accuracy and MS/MS spectra were validated manually using the Xcalibur™ software (Thermo Xcalibur 2.2 SP1.48 version).
Example 2: Novel High-Throughput Method for Discovery of Human MiHAs
Since MiHAs are i) peptides ii) whose presence depends on genetic polymorphisms, it was reasoned that high-throughput MiHA discovery would involve a combination of MS and personalized whole transcriptome and/or exome sequencing. Genomic data alone are insufficient for MiHA discovery because even if a genetic polymorphism is identified in a gene, i) only 0.1% of expressed peptides are presented as MAPs at the cell surface and ii) the effect of polymorphisms in trans cannot be predicted (5;6). In MS analyses, peptides are identified through database search with softwares such as Mascot. These databases contain the translated genome of reference and do not include genetic polymorphisms. Since MiHAs are the result of genetic polymorphisms, they cannot be discovered by standard MS alone.
A novel method for human MiHA discovery was developed. One of the key elements of the method is the inclusion of personalized translated transcriptome in the database used for peptide identification by MS.
PBMCs were isolated from blood samples of 2 HLA-identical siblings who are HLA-A*0301/2902, HLA-B*0801/*4403, HLA-C*0701/*1601. Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (B-LCLs) were derived from PBMCs with Ficoll-Paque™ Plus. Three biological replicates of 4×108 exponentially growing B-LCLs were prepared from each subject. MAPs were released by mild acid treatment and separated by cation exchange chromatography MAP fractions were analyzed by LC-MS/MS.
On each subject, a whole-exome and transcriptome sequencing was performed, and variants were identified. Based on the list of variants for each subject and human transcript annotations, a complete repertoire of subject-specific protein sequences was produced by translating annotated transcripts. While whole-exome sequencing provided a comprehensive identification of polymorphisms in coding regions, RNA-seq provided a most useful complementary dataset since i) it can highlight differences in transcript levels between two subjects (due for instance to polymorphisms in promoter regions), and ii) it covers unannotated genes or pseudogenes not covered by the exome capture protocol. The latter genes can be a source of MAPs and may have a significant contribution to the MiHA landscape. The translated exome-transcriptome were then used as a personalized Mascot protein sequence database, helping to retrieve MAPs overlapping variant locations and most importantly documenting differences in the MAP repertoire of HLA-identical siblings: a MAP present in only one of two HLA-identical siblings is an MiHA.
Example 3: Novel MiHAs Identified
Allelic MiHAs comprising the amino acid sequence ELQEKFLSL vs. ELQEKFSSL have been identified. These MiHAs are presented at the cell surface by the MHC class I allele HLA-B*0801. These peptides are not listed in the Immune Epitope Database (Vita et al., 2010) which includes a repertoire of all well characterized peptides presented by MHC molecules (HLA in humans). These MiHAs derive from a single nucleotide polymorphism in the centromer protein F, 350/400 kDa (mitosin) (CENPF) gene. This single nucleotide polymorphism is listed as rs3795517 in the dbSNP database (Sherry et al., 2001) (http://www.ncbi.nlm.nih.gov/projects/SNP/). Two alleles are found at this locus; one codes for a CENPF protein containing the ELQEKFLSL sequence, and the other codes for a CENPF protein with the ELQEKFSSL sequence. The single nucleotide polymorphism corresponds to a T to C substitution at a position corresponding to nucleotide 4409 in the nucleic acid sequence of human CENPF (FIGS. 1A to 1D, NCBI Reference Sequence: NM_016343.3), leading to a leucine to serine substitution at a position corresponding to residue 1412 in the CENPF protein sequence (FIG. 1E, NCBI Reference Sequence: NP_057427.3). CENPF is a transient kinetochore protein that plays multiple roles in cell division. CENPF expression increases in the G2 phase but it is rapidly proteolyzed at the end of mitosis. Overexpression of CENPF has been found in various hematopoietic cancers and solid tumors including head and neck squamous cell carcinomas (de la Guardia et al., 2001), breast cancer (O'Brien et al., 2007), non-Hodgkin's lymphoma (Bencimon et al., 2005) and gastrointestinal cancer (Chen et al., 2011). Also, according to the EMBL-EBI Gene Expression Atlas (Kapushesky et al., Nucl. Acids Res. (2012) (D1): D1077-D1081), overexpression of CENPF was detected in sarcomas (leiomyosarcomas), glioblastomas, lung adenocarcinomas, colorectal cancers/colon carcinomas, prostate cancer, bladder cancers, lymphomas (peripheral T-cell lymphomas) and leukemias (acute lymphocytic leukemias, T acute lymphoblastic leukemias). Accordingly, in an embodiment, the peptide of the sequence (I) described herein may be used in the immunotherapy of one or more of these cancers.
The amino acid sequence of another pair of MiHAs identified herein is QELDGVFQKL vs. QELDRVFQKL. These MiHAs are presented at the cell surface by HLA-B*4403 molecules. These peptides are not listed in the Immune Epitope Database (Vita et al., 2010). These MiHAs derive from a single nucleotide polymorphism in the ZWINT (ZW10 interactor) gene. This single nucleotide polymorphism is listed as rs2241666 in the dbSNP database (Sherry et al., 2001). Two alleles are found at this locus; one codes for a ZWINT protein containing the QELDGVFQKL sequence, and the other codes for a ZWINT protein with the QELDRVFQKL. sequence. The single nucleotide polymorphism corresponds to an A to G substitution at a position corresponding to nucleotide 596 in the nucleic acid sequence of human ZWINT (FIG. 2A, NCBI Reference Sequence: NM_007057.3), leading to an arginine to glycine substitution at a position corresponding to residue 187 in the ZWINT protein sequence (FIG. 2B, NCBI Reference Sequence: NP_008988.2). ZWINT is a known component of the kinetochore complex that is required for the mitotic spindle checkpoint. Overexpression of ZWINT has been observed in several types of cancer (notably breast, prostate and bladder) and correlates with an increased proliferation of cancer cells (Endo et al., 2012; Ho et al., 2012; Urbanucci et al., 2012). Also, according to the EMBL-EBI Gene Expression Atlas (Kapushesky et al., Nucl. Acids Res. (2012) 40 (D1): D1077-D1081), overexpression of ZWINT was detected in lung cancer (adenocarcinoma), head and neck squamous cell carcinoma, colorectal/colon cancer, renal carcinomas as well as lymphomas (mucosa-associated lymphoid tissue lymphomas, peripheral T-cell lymphomas). Accordingly, in an embodiment, the peptide of the sequence (II) described herein may be used in the immunotherapy of one or more of these cancers.
The amino acid sequence of a third pair of MiHAs identified herein is SLFFRKVPF vs. SLFFRKVAF. These MiHAs are presented at the cell surface by HLA-B*0801 molecules. These peptides are not listed in the Immune Epitope Database (Vita et al., 2010). These MiHAs derive from a single nucleotide polymorphism in the MTCH2 (mitochondrial carrier homologue 2) gene. This single nucleotide polymorphism is listed as rs1064608 in the dbSNP database (Sherry et al., 2001). Two alleles are found at this locus; one codes for a MTCH2 protein containing the SLFFRKVAF sequence, and the other codes for a MTCH2 protein with the SLFFRKVPF sequence. The single nucleotide polymorphism corresponds to a C to G substitution at a position corresponding to nucleotide 1057 in the nucleic acid sequence of human MTCH2 (FIG. 3A, NCBI Reference Sequence: NM_014342.3), leading to a proline to alanine substitution at a position corresponding to residue 290 in the MTCH2 protein sequence (FIG. 3B, NCBI Reference Sequence: NP_055157.1). MTCH2 interact with proapoptotic truncated BID and thereby regulate apoptosis, and is upregulated/involved in several types of cancer, notably solid tumors (lung, thyroid, liver, esophagus, colon, breast) and osteosarcoma (Yu et al., 2008, Grinberg et al., 2005; Katz et al., 2012). Also, according to the EMBL-EBI Gene Expression Atlas (Kapushesky et al., Nucl. Acids Res. (2012) 40 (D1): D1077-D1081), overexpression of MTCH2 was detected in lymphomas, leukemias and myelomas. Accordingly, in an embodiment, the peptide of the sequence (III) described herein may be used in the immunotherapy of one or more of these cancers.
The amino acid sequence of a fourth pair of MiHAs identified herein is SVLKPGNSK vs. TVLKPGNSK. These MiHAs are presented at the cell surface by HLA-A*0301 molecules. These peptides are not listed in the Immune Epitope Database (Vita et al., 2010). These MiHAs derive from a single nucleotide polymorphism in the ELF1 [E74-like factor 1 (ets domain transcription factor)] gene. This single nucleotide polymorphism is listed as rs1056820 in the dbSNP database (Sherry et al., 2001). Two alleles are found at this locus; one codes for an ELF1 protein containing the SVLKPGNSK sequence, and the other codes for an ELF1 protein with the TVLKPGNSK sequence. The single nucleotide polymorphism corresponds to an A to T substitution at a position corresponding to nucleotide 1400 in the nucleic acid sequence of human ELF1 (FIGS. 4A and 4B, NCBI Reference Sequence: NM_172373.3), leading to a threonine to serine substitution at a position corresponding to residue 343 in the ELF1 protein sequence (FIG. 4C, NCBI Reference Sequence: NP_758961.1). ELF1 is a transcriptional factor that is involved in the transcriptional activation of oncogenic pathways in cancer cells, such as leukemias, non-small cell lung cancer, breast and ovarian cancer (Andrews et al., 2008; Xiang et al., 2010; Yang et al., 2010). Also, according to the EMBL-EBI Gene Expression Atlas (Kapushesky et al., Nucl. Acids Res. (2012) 40 (D1): D1077-D1081), overexpression of ELF1 was detected in sarcomas (e.g., osteosarcomas), glioblastomas, pancreatic cancer and leukemias (acute myeloid leukemia), lymphomas (Burkitt's lymphomas). Accordingly, in an embodiment, the peptide of the sequence (IV) described herein may be used in the immunotherapy of one or more of these cancers.
The amino acid sequence of another pair of MiHAs is AMYDKGPFRSK vs. AMYDKGPFWSK. These MiHAs are presented at the cell surface by HLA-A*0301. These peptides are not listed in the Immune Epitope Database (Vita et al., 2010). These MiHAs derive from a single nucleotide polymorphism in the NQO1 [NAD(P)H dehydrogenase, quinone 1] gene. This single nucleotide polymorphism is listed as r51131341 in the dbSNP database (Sherry et al., 2001). Two alleles are found at this locus; one codes for an NQO1 protein containing the AMYDKGPFRSK sequence, and the other codes for an NQO1 protein with the AMYDKGPFWSK sequence. The single nucleotide polymorphism corresponds to a C to T substitution at a position corresponding to nucleotide 615 in the nucleic acid sequence of human NQO1 (FIG. 5A, NCBI Reference Sequence: NM_000903.2), leading to an arginine to tryptophan substitution at a position corresponding to residue 139 in the NQO1 protein sequence (FIG. 5B, NCBI Reference Sequence: NP_000894.1). NQO1 is a cytosolic enzyme that catalyzes the reduction of various quinones using flavin adenine dinucleotide (FAD) as a cofactor. This protein's enzymatic activity prevents the one electron reduction of quinones that results in the production of radical species. Mutations in this NQO1 have been associated with susceptibility to various forms of cancer and altered expression of this NQO1 protein has been seen in many tumors, including non-small cell lung cancer, skin cancer (e.g., melanomas), breast cancer, liver cancer (e.g., intrahepatic cholangiocarcinoma) and digestive tract cancer such as colorectal cancer (Kolesar et al., 2011; Wakai et al., 2011; Jamieson et al., 2011; Ding et al., 2012; Yang et al., 2012; Patrick and Jaiswal, 2012). An NQO1 Substrate was recently shown to possess potent antitumor activity against a wide spectrum of cancer cells, such as pancreatic and lung cancer cells (Huang et al., 2012). Also, according to the EMBL-EBI Gene Expression Atlas (Kapushesky et al., Nucl. Acids Res. (2012) 40 (D1): D1077-D1081), overexpression of NQO1 was detected in lymphomas (Hodgkin's lymphomas, anaplastic large cell lymphoma) and brain cancers (glioblastomas, subependymal giant cell astrocytomas). Accordingly, in an embodiment, the peptide of the sequence (V) described herein may be used in the immunotherapy of one or more of these cancers.
The amino acid sequence of another pair of MiHAs is RVSLPTSPG vs. RVSLPTSPR. These MiHAs are presented at the cell surface by HLA-A*0301. These peptides are not listed in the Immune Epitope Database (Vita et al., 2010) which includes a repertoire of all well characterized peptides presented by MHC molecules (HLA in humans).
These MiHAs derive from a single nucleotide polymorphism in the KIAA0226-like gene (also known as C13orf18). This single nucleotide polymorphism is listed as rs1408184 in the dbSNP database (Sherry et al., 2001). Two alleles are found at this locus; one codes for an KIAA0226L protein containing the RVSLPTSPG sequence, and the other codes for an KIAA0226L protein with the RVSLPTSPR sequence. The single nucleotide polymorphism corresponds to a G to A substitution at a position corresponding to nucleotide 1059 in the nucleic acid sequence of human KIAA0226L (FIG. 6A, NCBI Reference Sequence: NM_025113.2), leading to a glycine to arginine substitution at a position corresponding to residue 152 in the KIAA0226L protein sequence (FIG. 6B, NCBI Reference Sequence: NP_079389.2). The function of KIAA0226L is largely unknown, and according to the BioGPS database, KIAA0226L is upregulated in B lymphocytes and Burkitt's lymphoma cells. Also, according to the EMBL-EBI Gene Expression Atlas (Kapushesky et al., Nucl. Acids Res. (2012) 40 (D1): D1077-D1081), overexpression of KIAA0226L was detected in colon cancer (carcinoma), glioblastoma, and anaplastic large cell lymphoma (Chowdary D et al., J Mol Diagn. 2006 February; 8(1):31-9; Ancona N et al., BMC Bioinformatics. 2006 Aug. 19; 7:387; Freije W A et al., Cancer Res. 2004 Sep. 15; 64(18):6503-10; Sun et al., Cancer Cell. 2006 April; 9(4):287-300; Piccaluga et al., J Clin Invest. 2007 March; 117(3):823-34. Epub 2007 Feb. 15). Accordingly, in an embodiment, the peptide of the sequence (VI) described herein may be used in the immunotherapy of one or more of these cancers.
The amino acid sequence of another pair of MiHAs is VMGNPGTFK vs. VMGNPGTFN. These MiHAs are presented at the cell surface by HLA-A*0301. These peptides are not listed in the Immune Epitope Database (Vita et al., 2010) which includes a repertoire of all well characterized peptides presented by MHC molecules (HLA in humans).
These MiHAs derive from a single nucleotide polymorphism in the RMDN1 (regulator of microtubule dynamics 1) gene (also known as FAM82B). This single nucleotide polymorphism is listed as rs6980476 in the dbSNP database (Sherry et al., 2001). Two alleles are found at this locus; one codes for an RMDN1 protein containing the VMGNPGTFK sequence, and the other codes for an RMDN1 protein with the VMGNPGTFN sequence. The single nucleotide polymorphism corresponds to an A to C substitution at a position corresponding to nucleotide 316 in the nucleic acid sequence of human RMDN1 (FIG. 7A, NCBI Reference Sequence: NM_016033.2), leading to a lysine to asparagine substitution at a position corresponding to residue 52 in the RMDN1 protein sequence (FIG. 7B, NCBI Reference Sequence: NP_057117.2). RMDN1 is a microtubule-associated protein that plays a role in chromosome segregation in Caenorhabditis elegans (Oishi et al., J Cell Biol. 179, 1149-1162, 2007). According to the BioGPS database, RMDN1 is upregulated in B lymphoblasts and Burkitt's lymphoma cells. Also, according to the EMBL-EBI Gene Expression Atlas (Kapushesky et al., Nucl. Acids Res. (2012) 40 (D1): D1077-D1081), overexpression of RMDN1 was detected in leiomyosarcoma (Perot et al., Cancer Res. 2009 Mar. 15; 69(6):2269-78; Chibon et al., Nat Med. 2010 July; 16(7):781-7), breast cancer/carcinoma such as invasive ductal carcinoma (Chen et al., Breast Cancer Res Treat. 2010 January; 119(2):335-46; Cheng et al., Cancer Res. 2008 Mar. 15; 68(6):1786-96), and brain cancers such as glioblastomas and astrocytomas (subependymal giant cell astrocytomas (Sun et al., Cancer Cell. 2006 April; 9(4):287-300; Tyburczy et al., Am J Pathol. 2010 April; 176(4):1878-90). Accordingly, in an embodiment, the peptide of the sequence (VII) described herein may be used in the immunotherapy of one or more of these cancers.
Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims. In the claims, the word “comprising” is used as an open-ended term, substantially equivalent to the phrase “including, but not limited to”. The singular forms “a”, “an” and “the” include corresponding plural references unless the context clearly dictates otherwise.
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Claims (10)

What is claimed is:
1. A method of treating cancer, said method comprising administering to a subject in need thereof an effective amount of CD8 T lymphocytes recognizing:
(a) a MHC class I molecule of the HLA-B*0801 allele loaded with a peptide of 8 to 12 amino acids comprising (i) one of the following sequences: ELQEKFLSL (SEQ ID NO:15) or ELQEKFSSL (SEQ ID NO:16); and/or (ii) one of the following sequences: SLFFRKVPF (SEQ ID NO:19) or SLFFRKVAF (SEQ ID NO:20), if said subject expresses said MHC class I molecule of the HLA-B*0801 allele;
(b) a MHC class I molecule of the HLA-B*4403 allele loaded with a peptide of 10 to 12 amino acids comprising one the following sequences: QELDGVFQKL (SEQ ID NO:17) or QELDRVFQKL (SEQ ID NO:18), if said subject expresses said MHC class I molecule of the HLA-B*4403 allele; and/or
(c) a MHC class I molecule of the HLA-A*0301 allele loaded with a peptide of 9 to 12 amino acids comprising (i) one of the following sequences: RVSLPTSPG (SEQ ID NO:25) or RVSLPTSPR (SEQ ID NO:26); (ii) one of the following sequences: SVLKPGNSK (SEQ ID NO:21) or TVLKPGNSK (SEQ ID NO:22); (iii) one of the following sequences: AMYDKGPFRSK (SEQ ID NO:23) or AMYDKGPFWSK (SEQ ID NO:24); and/or (iv) one of the following sequences: VMGNPGTFK (SEQ ID NO:27) or VMGNPGTFN (SEQ ID NO:28), if said subject expresses said MHC class I molecule of the HLA-A*0301 allele.
2. The method of claim 1, wherein said CD8 T lymphocytes are in vitro expanded primary CD8 T lymphocytes or CD8 T lymphocyte clones expressing a recombinant T cell receptor (TCR).
3. The method of claim 1, wherein said subject is an allogeneic stem cell transplantation (ASCT) recipient.
4. The method of claim 1, wherein said cancer is a hematologic cancer.
5. The method of claim 1, wherein said method further comprises administering an effective amount of one or more of the peptides defined in claim 1, or of a pharmaceutical composition comprising said one or more peptides.
6. The method of claim 1, wherein said method further comprises culturing and expanding said CD8 T lymphocytes in the presence of cells expressing said MHC class I molecule loaded with said peptide in vitro prior to administration to the subject.
7. The method of claim 6, wherein said CD8 T lymphocytes are in vitro expanded primary CD8 T lymphocytes or CD8 T lymphocyte clones expressing a recombinant T cell receptor (TCR).
8. The method of claim 6, wherein said subject is an allogeneic stem cell transplantation (ASCT) recipient.
9. The method of claim 6, wherein said cancer is a hematologic cancer.
10. The method of claim 6, wherein said method further comprises administering an effective amount of one or more of the peptides defined in claim 1, or of a pharmaceutical composition comprising said one or more peptides.
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